Temperature-responsive polymer compound and process for producing the same

ABSTRACT

A temperature-responsive polymer and polymer material which has ester bond(s) and/or acid amide bond(s) respectively at one or more sites in the side chain and can be arbitrarily controlled by varying the side chain is provided. The process for production thereof and the use thereof are also provided.

TECHNICAL FIELD

This invention relates to a novel polymer compound which undergoesstretching and cohesion with a change in the polarity of the polymer perse due to a temperature change, a process for producing this polymercompound, a heat-responsive polymer material containing this compound, aseparation method with the use of a material containing thisheat-responsive polymer material, and a method for separating chemicals,biological polymers (proteins, peptides, etc.) and biological samples(cells, etc.) by using this material.

In addition, this invention relates to a temperature-responsive polymercompound a change in the characteristics of which can be controlleddepending on temperature. Furthermore, the present invention relates toa temperature-responsive polymer compound which is usable, by takingadvantage of the temperature-depending change in the characteristicsthereof, in adsorption and separation materials, drug carriers,dielectric and magnetic materials, piezoelectric and pyroelectricmaterials, degradable and reactive materials, biofunctional materials,etc.

This invention also relates to a temperature-responsive polymer compoundby which the polarity and hydrogen-bonding ability of a material can bechanged or controlled depending on temperature. The present inventionfurther relates to a temperature-responsive polymer compound which isusable in materials for adsorbing, separating and releasing substancesto be applied to biological functions with the use of a change in thepolarity and the hydrogen-bonding ability depending on temperature. Thepresent invention furthermore relates to a method for adsorbing,separating, recovering and releasing substances characterized by usingthese polymer compounds.

It should be noted that the terms “heat-responsive” and“temperature-responsive” used herein have the same meaning.

BACKGROUND ART

Typical examples of heat-responsive polymer materials having ester bondsor acid amide bonds include partially oxidized polyvinyl alcohol andN-isopropyl acrylamides. It is known that the cloud point of an esterbond-type polymer or an alkylamide polymer would be gradually loweredwith an increase in the carbon atom number in a side chain. It istherefore impossible to synthesize a heat-responsive polymer of thealkylamide type having a side chain with a large carbon atom number. Inthe case of an ester-bond type polymer or an alkylamide polymer, it isalso difficult to provide a sufficient polarity in separating anyprotein.

N-Alkylacrylamides typified by N-isopropylacrylamide, which in polymerform are known to be temperature-responsive, have been frequentlyapplied to DDS (Drug Delivery System) and separating agents. However,alkylacrylamide monomers showing temperature-responsiveness in polymerform carry exclusively alkyl groups with a small number of carbon atoms.Owing to this characteristic, these alkylacrylamides are poor in thehydrophobic nature or the hydrogen bonding properties, which makes itdifficult to efficiently separate, adsorb and release all biologicalcomponents or organic matters by using the these kinds of polymers.Although hydrogen-bonding groups can be introduced into these compoundsby forming a copolymer with the use of a monomer having ahydrogen-bonding group, there arises a problem in this case, i.e., anincrease in the cloud point or the disappearance of thetemperature-responsiveness. As a result, a target substance, inparticular, a biological component should be separated, adsorbed andreleased under severe conditions.

In polymer compounds showing structural changes due to external stimuli(temperature, pH, light, etc.), the structural changes result in changesin the characteristics of the polymers, for example, volume orhydrophilic/hydrophobic nature. For example, it is well known thatpoly(N-isopropyl acrylamide) shows a structural change in an aqueoussolution depending on temperature. Namely, this compound is soluble inwater in a low temperature side of 32° C. or below but becomes insolublein water in a high temperature side exceeding 32° C. That is to say, itis a temperature-responsive polymer compound having a lower criticalsolution temperature (LCST). It is considered that such a polymercompound would show a hydrophilic nature and be dissolved in water in aswollen state in the low temperature side and, in the high temperatureside, it would show a hydrophobic nature and be aggregated in acontracted state. By using these temperature-depending changes,temperature-responsive polymer compounds have been applied to drugdelivery systems and high-functional materials such as separators.

To apply these temperature-responsive polymer compounds tohigh-functional materials, it is needed to use not onlytemperature-responsive polymer compounds having LCST but also thosehaving the upper limit critical solution temperature (UCST), i.e., beinginsoluble in water in a low temperature side but becoming soluble inwater in a high temperature side. When a protein unstable to heat is tobe separated by adsorbing on a temperature-responsive polymer compoundvia a hydrophobic action, a temperature-responsive polymer compoundhaving the UCST, i.e., showing a hydrophobic nature at low temperatures,is seemingly useful. At present, however, there are knowntemperature-responsive polymer compounds of few types having the UCSTand it is difficult to newly develop temperature-responsive polymercompounds usable as efficient high-functional materials.

To obtain high-functional temperature-responsive polymer compounds, itis necessary to develop temperature-responsive polymer compounds havingnovel characteristics which are not achieved by the existing ones. Ingeneral, it is considered that a temperature-responsiveness is expressedowing to the balance between a hydrophilic moiety and a hydrophobicmoiety. For example, a temperature-responsive polymer compound becomesinsoluble in water with an increase in the carbon atom number in theside chain thereof. It is, therefore, difficult to synthesize atemperature-responsive polymer compound having a side chain with a largecarbon atom number.

DISCLOSURE OF INVENTION

Under these circumstances, an object of the present invention is toprovide a heat-responsive polymer material having a side chain with alarge carbon atom number and showing various polarities which issynthesized by introducing acid amide bond(s) and ester bond(s)respectively into a side chain at one or more sites in the polymerchain, and a process for producing the same. Another object of thepresent invention is to apply the above-mentioned heat-responsivepolymer to the separation and purification of proteins, chemicals orbiological samples such as bioengineering products and cells havingvarious polarities.

Another object of the present invention is to provide a polymer compoundthe temperature-responsiveness of which can be controlled by changingthe functional groups or composition of the monomers constituting thepolymer. Another object of the present invention is to provide atemperature-responsive polymer compound having an aromatic ring andbeing expected as exerting a high hydrophobicity or an electronicinteraction which cannot be achieved by the existingtemperature-responsive polymer compounds. A still further object of thepresent invention is to provide separation materials such aschromatographic packings containing these temperature-responsive polymercompounds.

A further object of the present invention is to provide aheat-responsive polymer which has a side chain with a large carbonnumber capable of imparting a hydrophobic nature thereto together with afunctional group having hydrogen-bonding properties.

The present invention aims at providing a material which undergoesinteraction with substances including biological components with the useof the hydrogen-bonding properties and hydrophobic nature thereof andcan show the LCST or the UCST even in an aqueous solution containing asalt. The present invention further aims at providing novel materials(chromatographic packings, etc.) for the separation, adsorption orrelease of substances with the use of the above-described polymer.

To synthesize a heat-responsive polymer material having a side chainwith a large carbon atom number, the present inventors have produced amaterial having a side chain with a large carbon atom number byintroducing acid amide bond(s) and ester bond(s) respectively into oneor more sites in the side chain. Then they have found that the thusobtained material shows a heat-responsiveness and various polarities.The present inventors have further found that this material isapplicable to the separation of bioengineering products (proteins,peptides, etc.) having various polarities. The present invention hasbeen completed based on these findings.

The present inventors have conducted intensive studies and consequentlyfound that a temperature-responsive polymer compound thetemperature-responsiveness of which can be controlled can be synthesizedby appropriately specifying functional groups of the monomer to bepolymerized. The present inventors have further found that atemperature-responsive polymer compound the temperature-responsivenessof which can be controlled can be synthesized by appropriatelyspecifying the functional groups or composition of two types of monomersto be copolymerized. The present inventors have furthermore found thatadsorption and separation materials containing thesetemperature-responsive polymer compounds are applicable to theseparation of various substances. The present invention has beencompleted based on these findings.

To obtain heat-responsive polymers having hydrogen-bonding propertiesand highly hydrophobic nature, the present inventors have synthesizedhydroxyalkylamide monomers with a large carbon atom number in theiralkyl groups and polymerized the same to give polymer materials havingboth of hydrogen-bonding groups and hydrophobic groups. They have foundthat these polymer materials show temperature-responsiveness. Thepresent inventors have further found that these temperature-responsivepolymer compounds are usable in materials for separating, adsorbing andreleasing substances and thus various substances can be separated byapplying these materials. The present invention has also been completedbased on these findings.

Further, the present inventors have conducted intensive studies andconsequently found that a temperature-responsive polymer compound, thetemperature-responsiveness of which can be controlled, can besynthesized by introducing a hydrogen-bonding functional group and ahydrophobic group into a monomer followed by polymerization, or bycopolymerizing such a monomer with another polymerizable monomer. Thepresent inventors have also found that adsorption/separation materialscontaining the above temperature-responsive polymer compound areapplicable to the separation of various substances. The presentinvention has been completed based on these findings.

Accordingly, the present invention provides a polymer compoundcomprising polymer subunits as defined in groups A-E and with the totalor relative number of individual monomer units as given.

wherein R represents a linear or branched divalent aliphatic hydrocarbongroup having 1 to 8 carbon atoms, a divalent alicyclic hydrocarbon grouphaving 3 to 8 carbon atoms, or a divalent aromatic hydrocarbon grouphaving 6 to 14 carbon atoms; R′ represents a linear or branchedaliphatic hydrocarbon group having 1 to 8 carbon atoms, a linear orbranched aliphatic hydrocarbon group having one or more hydroxyl groupsand 1 to 8 carbon atoms, a linear or branched aliphatic hydrocarbongroup having one or more acid amide bonds and/or ester bonds and 2 to 9carbon atoms, or a linear or branched aliphatic hydrocarbon group havingone or more acid amide bonds and/or ester bonds, one or more hydroxylgroups and 3 to 9 carbon atoms; and n is an integer of 2 or above.

wherein R represents a linear or branched divalent aliphatic hydrocarbongroup having 1 to 8 carbon atoms, a divalent alicyclic hydrocarbon grouphaving 3 to 8 carbon atoms, or a divalent aromatic hydrocarbon grouphaving 6 to 14 carbon atoms; R′ represents a linear or branchedaliphatic hydrocarbon group having 1 to 8 carbon atoms, a linear orbranched aliphatic hydrocarbon group having one or more hydroxyl groupsand 1 to 8 carbon atoms, a linear or branched aliphatic hydrocarbongroup having one or more acid amide bonds and/or ester bonds and 2 to 9carbon atoms, or a linear or branched aliphatic hydrocarbon group havingone or more acid amide bonds and/or ester bonds, one or more hydroxylgroups and 3 to 9 carbon atoms; and n is an integer of 2 or above.

wherein n is the number of the middle kind of monomer unit, j is thenumber of the right kind of monomer unit, n is from 0.005 to 0.995(inclusive) and j is from 0 to 0.5 (inclusive); R¹, R², R³ and R⁴ arethe same or different and each represents a hydrogen atom or a methylgroup; X¹, X², X³ and X⁴ are the same or different and each representsan acid amide group, an ester group or an ether group; Y¹ represents alinear or branched divalent aliphatic hydrocarbon group having 1 to 8carbon atoms, a divalent alicyclic hydrocarbon group having 3 to 8carbon atoms or a divalent aromatic hydrocarbon group having 6 to 14carbon atoms; Y² represents a linear or branched divalent aliphatichydrocarbon group having 1 to 8 carbon atoms, a divalent alicyclichydrocarbon group having 3 to 8 carbon atoms, a linear or brancheddivalent aliphatic hydrocarbon group having 1 to 8 carbon atoms and oneor more ether groups or a linear or branched divalent aliphatichydrocarbon group having 1 to 8 carbon atoms and one or more hydroxylgroups; Z¹, Z², Z³, Z⁵ and Z⁶ are the same or different and eachrepresents a hydrogen atom, a linear or branched aliphatic hydrocarbongroup having 1 to 8 carbon atoms, a linear or branched aliphatichydrocarbon group having 1 to 8 carbon atoms and one or more hydroxylgroups, a linear or branched alicyclic hydrocarbon group having 1 to 8carbon atoms and one or more hydroxyl group, a linear or branchedaliphatic hydrocarbon group having 1 to 8 carbon atoms and one or moreether groups, a linear or branched alicyclic hydrocarbon group having 1to 8 carbon atoms and one or more ether groups, a glycoside having 3 to12 carbon atoms or a glycoside having 3 to 12 carbon atoms and carryinga linear or branched aliphatic hydrocarbon group having 1 to 8 carbonatoms, provided that Z¹, Z³, Z⁵ and Z⁶ are functional groups bondedrespectively to X¹, X², X³ and X⁴ when they are tertiary amide groupsand Z⁵ may be bonded to Z⁶; and Z⁴ represents a hydrogen atom, ahydroxyl group, an amide group, a nitryl group, a linear or branchedaliphatic hydrocarbon group having 1 to 8 carbon atoms, a linear orbranched aliphatic hydrocarbon group having 1 to 8 carbon atoms and oneor more amide groups, a linear or branched aliphatic hydrocarbon grouphaving 1 to 8 carbon atoms and one or more carbonyl groups, a linear orbranched aliphatic hydrocarbon group having 1 to 8 carbon atoms and oneor more nitryl groups, or a linear or branched aliphatic hydrocarbongroup having 1 to 8 carbon atoms and one or more hydroxyl groups.

wherein R⁵ represents a hydrogen atom or a methyl group; X⁵ representsan acid amide group, an ester group or an ether group; Y³ represents alinear or branched divalent aliphatic hydrocarbon group having 1 to 8carbon atoms or a divalent alicyclic hydrocarbon group having 3 to 8carbon atoms; and Y⁴ represents a linear or branched aliphatichydrocarbon group having 1 to 8 carbon atoms or a linear or branchedaliphatic hydrocarbon group having 1 to 8 carbon atoms and one or morehydroxyl groups; n represent an integer 2 or more.

wherein Z represents a hydrogen atom or a methyl group; X represents ahydrogen atom or a linear or branched aliphatic hydrocarbon group having1 to 8 carbon atoms and carrying at least one hydroxyl group; Yrepresents a linear or branched aliphatic hydrocarbon group having 2 to8 carbon atoms and carrying at least one hydroxyl group, or X and Y mayform together a chemical bond; and n is an integer of 2 or more.

wherein Z represents a methyl group or a hydrogen atom; n is an integerof 2 or more; X₁, X₂, X₃, X₄ and X₅ are the same or different and eachrepresents a hydrogen atom, a group R, or a group —CO—NH—R, providedthat at least one of X₁ to X₅ is a group —CO—NH—R (wherein R representsa linear or branched aliphatic hydrocarbon group having 1 to 6 carbonatoms, a linear or branched aliphatic hydrocarbon group having 1 to 10carbon atoms and containing at least one amide bond, a linear orbranched aliphatic hydrocarbon group having 1 to 10 carbon atoms andcontaining at least one hydroxyl group, an alicyclic hydrocarbon grouphaving 3 to 10 carbon atoms and containing at least one hydroxyl group,an alicyclic hydrocarbon group having 3 to 10 carbon atoms andcontaining at least one amide bond, or a hydrogen atom); and i is aninteger of from 0 to 6.

wherein n is the number of the left kind of monomer unit and m is thenumber of the right kind of monomer unit compared to the sum of themwith n+m=1.0; Z represents a methyl group or a hydrogen atom; X₁, X₂,X₃, X₄ and X₅ are the same or different and each represents a hydrogenatom, a group R, or a group —CO—NH—R, provided that at least one of X₁to X₅ is a group —CO—NH—R (wherein R represents a linear or branchedaliphatic hydrocarbon group having 1 to 6 carbon atoms, a linear orbranched aliphatic hydrocarbon group having 1 to 10 carbon atoms andcontaining at least one amide bond, a linear or branched aliphatichydrocarbon group having 1 to 10 carbon atoms and containing at leastone hydroxyl group, an alicyclic hydrocarbon group having 3 to 10 carbonatoms and containing at least one hydroxyl group, an alicyclichydrocarbon group having 3 to 10 carbon atoms and containing at leastone amide bond, or a hydrogen atom); i is an integer of from 0 to 6; Yrepresents an oxygen atom or a nitrogen atom; and R′ represents a linearor branched aliphatic hydrocarbon group having 1 to 6 carbon atoms, alinear or branched aliphatic hydrocarbon group having 1 to 10 carbonatoms and containing at least one amide bond, a linear or branchedaliphatic hydrocarbon group having 1 to 10 carbon atoms and containingat least one hydroxyl group, an alicyclic hydrocarbon group having 3 to10 carbon atoms and containing at least one hydroxyl group, an alicyclichydrocarbon group having 3 to 10 carbon atoms and containing at leastone amide bond or a hydrogen atom.

wherein Z represents a methyl group or a hydrogen atom; n is an integerof 2 or more; X₁, X₂, X₃, X₄ and X₅ are the same or different and eachrepresents a hydrogen atom, a group R, or a group —CO—NH—R, providedthat at least one of X₁ to X₅ is a group —CO—NH—R (wherein R representsa linear or branched aliphatic hydrocarbon group having 1 to 6 carbonatoms, a linear or branched aliphatic hydrocarbon group having 1 to 10carbon atoms and containing at least one amide bond, a linear orbranched aliphatic hydrocarbon group having 1 to 10 carbon atoms andcontaining at least one hydroxyl group, an alicyclic hydrocarbon grouphaving 3 to 10 carbon atoms and containing at least one hydroxyl group,an alicyclic hydrocarbon group having 3 to 10 carbon atoms andcontaining at least one amide bond, or a hydrogen atom); A₁, A₂, A₃, A₄and A₅ are the same or different and each represents a carbon atom or anitrogen atom bonding to X_(n) (wherein n is an integer of 1 to 5)having a group —CO—NH—R or a group —CO—R, provided that at least one ofA₁ to A₅ is a nitrogen atom bonding to X_(n) (wherein n is an integer of1 to 5) having a group —CO—NH—R or a group —CO—R (wherein R is asdefined above); and i is an integer of from 0 to 6.

wherein n is the number of the left kind of monomer unit and m is thenumber of the right kind of monomer unit compared to the sum of them andwith n+m=1.0; Z represents a methyl group or a hydrogen atom; X₁, X₂,X₃, X₄ and X₅ are the same or different and each represents a hydrogenatom, a group R, or a group —CO—NH—R, provided that at least one of X₁to X₅ is a group —CO—NH—R (wherein R represents a linear or branchedaliphatic hydrocarbon group having 1 to 6 carbon atoms, a linear orbranched aliphatic hydrocarbon group having 1 to 10 carbon atoms andcontaining at least one amide bond, a linear or branched aliphatichydrocarbon group having 1 to 10 carbon atoms and containing at leastone hydroxyl group, an alicyclic hydrocarbon group having 3 to 10 carbonatoms and containing at least one hydroxyl group, an alicyclichydrocarbon group having 3 to 10 carbon atoms and containing at leastone amide bond, or a hydrogen atom); A₁, A₂, A₃, A₄ and A₅ are the sameor different and each represents a carbon atom or a nitrogen atombonding to X_(n) (wherein n is an integer of 1 to 5) having a group—CO—NH—R or a group —CO—R, provided that at least one of A₁ to A₅ is anitrogen atom bonding to X_(n) (wherein n is an integer of 1 to 5)having a group —CO—NH—R or a group —CO—R (wherein R is as definedabove); i is an integer of from 0 to 6; Y represents an oxygen atom or anitrogen atom; and R′ represents a linear or branched aliphatichydrocarbon group having 1 to 6 carbon atoms, a linear or branchedaliphatic hydrocarbon group having 1 to 10 carbon atoms and containingat least one amide bond, a linear or branched aliphatic hydrocarbongroup having 1 to 10 carbon atoms and containing at least one hydroxylgroup, an alicyclic hydrocarbon group having 3 to 10 carbon atoms andcontaining at least one hydroxyl group, an alicyclic hydrocarbon grouphaving 3 to 10 carbon atoms and containing at least one amide bond or ahydrogen atom.

wherein n is the number of the right kind of monomer unit compared tothe total number of the two kinds of monomer units shown and is anarbitrary value falling within the range 0.005≦n≦0.995; R¹ and R² arethe same or different and each represents a hydrogen atom or a methylgroup; X¹ and X² are the same or different and each represents an acidamide or ester group; Z¹, Z² and Z³ are the same or different and eachrepresents a hydrogen atom, a linear or branched aliphatic hydrocarbongroup having 1 to 8 carbon atoms, a linear or branched hydrocarbon grouphaving 1 to 8 carbon atoms and containing at least one hydroxyl group, alinear or branched hydrocarbon group having 1 to 8 carbon atoms andcontaining at least one ether group, a glycoside having 3 to 12 carbonatoms or a glycoside having 3 to 12 carbon atoms and containing a linearor branched hydrocarbon group having 1 to 8 carbon atoms, provided thatZ¹ or Z³ is a functional group carried by X¹ or X² which is an acidamide; and Z⁴ represents a hydrogen atom, a hydroxyl group, an amidegroup, a linear or branched hydrocarbon group having 1 to 8 carbon atomsand containing at least one amide group, a linear or branchedhydrocarbon group having 1 to 8 carbon atoms and containing at least onecarbonyl group or a linear or branched hydrocarbon group having 1 to 8carbon atoms and containing at least one hydroxyl group which may beattached at an arbitrary position, i.e., o-, m- or p-position.

The present invention further provides a heat-responsive polymermaterial which contains a polymer compound represented by the formula ofGroup A and shows a cloud point due to a temperature change in anaqueous solution.

The present invention further provides a chromatographic packing with astationary phase containing a heat-responsive polymer material whichcontains a polymer compound represented by the formula of Group A andshows a cloud point due to a temperature change in an aqueous solution.In addition, the present invention further provides a method forseparating substances characterized by comprising the steps:

-   -   (i) adsorbing/binding a substance to a stationary phase present        on the chromatographic packing as described above,    -   (ii) changing the hydrophilic/hydrophobic balance of the surface        of the stationary phase by changing the temperature, preferably        by external means and preferably in one or more steps,    -   (iii) passing a mobile phase, preferably having a constant        composition and preferably being a liquid through the        chromatographic packing;        steps (ii) and (iii) effecting release and separation of the        substance from the packing. The present invention furthermore        provides a process for producing a polymer compound represented        by the formula of Group A characterized by using one of the        following methods:    -   (1) reacting an acrylic/methacryl amide or acrylic/methacryl        ester monomer having a primary amino group in its amine or ester        part, respectively, (for example, 2-aminoethyl methacrylate)        with an acid anhydride or lactone and purifying the thus        obtained product followed by polymerization in a solvent;    -   (2) reacting an acrylic/methacryl amide or acrylic/methacryl        ester monomer having a hydroxyl group in its amine or ester part        with an acid chloride and purifying the thus obtained product        followed by polymerization in a solvent;    -   (3) reacting an alkylamino alcohol with an acid anhydride, then        reacting the thus obtained product with acrylic acid chloride or        methacrylic acid chloride and purifying the thus obtained        product followed by polymerization in a solvent; or    -   (4) synthesizing a poly(acryl/methacryl amide or ester) having a        primary amino group (for example, poly-2-aminoethyl        methacrylate) or its hydrochloride in its amino or alcohol part,        respectively, and reacting the thus synthesized product with an        acid anhydride or lactone in a solvent containing triethylamine.

The present invention further provides a material for separating oradsorbing biological samples comprising a polymer compound representedby the formula of Group A and having acid amide bonds at two or moresites in the polymer side chain.

In addition, the present invention provides a method for separatingsubstances characterized by comprising the steps:

-   -   (i) adsorbing/binding a substance, for instance from a        biological sample, to a stationary phase present on a        chromatographic packing,    -   (ii) changing the hydrophilic/hydrophobic balance of the        stationary phase by changing the temperature of the stationary        phase, preferably by external means and preferably in one or        more steps,    -   (iii) passing a mobile phase through the packing, preferably of        essentially constant composition and preferably in liquid form;        steps (i) and (ii) effecting release and separation of the        substance from the stationary phase, and wherein said stationary        phase comprises a polymer compound represented by the formula of        Group A and having amide bonds in the side chain at two or more        sites in the polymer chain.

The present invention further provides a heat-responsive polymermaterial which contains a polymer compound represented by the formula ofGroup B and shows a cloud point due to a temperature change in anaqueous solution.

The present invention further provides a chromatographicpacking/stationary phase containing a heat-responsive polymer materialwhich contains a polymer compound represented by the formula of Group Band shows a cloud point due to a temperature change in an aqueoussolution.

In addition, the present invention provides a method for separatingsubstances characterized by comprising:

-   -   (i) binding/adsorbing a substance to a stationary phase on a        chromatographic packing as described above,    -   (ii) changing the hydrophilic/hydrophobic balance of the surface        of the stationary phase by changing the temperature, preferably        by external means and preferably in one or more steps, and    -   (iii) passing single mobile phase through the packing,        steps (ii) and (iii) effecting release and separation of the        substance from the stationary phase. In this aspect of the        invention the stationary phase comprises a polymer compound        complying with a member of Group B above.

The present invention furthermore provides a process for producing apolymer compound represented by the formula of Group B characterized byusing one of the following methods:

-   -   (1) reacting a compound selected from among aminoalkyl        acylamide, aminoalkyl methacrylamide, aminoalkyl acrylamide        hydrochloride and aminoalkyl methacrylamide hydrochloride with        an acid anhydride or lactone, and purifying the thus obtained        product followed by polymerization in a solvent; and    -   (2) reacting an alkyl diamine with an acid anhydride or an        alkyll acid chloride, or reacting a compound having an amino        group and an amide bond in its molecule with acryloyl chloride        or methacryloyl chloride, and then purifying the thus obtained        product followed by polymerization in a solvent.

The present invention further provides a material for separating oradsorbing biological samples comprising a polymer compound representedby the formula of Group B and having acid amide bonds at two or moresites in the polymer side chain.

In addition, the present invention provides a method as just definedabove. In this aspect the stationary phase comprises a polymer compoundrepresented by the formula of Group B and having acid amide bonds in theside chain at two or more sites in the polymer chain.

The present invention furthermore provides the above-mentioned polymercompound of Group C-1 or Group C-2 characterized by containing anaromatic hydrocarbon group and said repeating unit of the polymercontaining two or more amide or ester groups which are either the sameor different.

The present invention furthermore provides a polymer compound selectedform the group consisting of the polymers represented by the formula ofGroup C-1 or Group C-2 and crosslinked matters containing these polymerscharacterized by expressing a temperature-responsiveness of changing itscharacteristics under a temperature change.

In addition, the present invention provides the above-mentionedtemperature-responsive polymer compound of Group C-1 or Group C-2characterized by being obtained by copolymerizing monomers which do notexpress any temperature-responsiveness each as a homopolymer.

Moreover, the present invention provides the above-mentionedtemperature-responsive polymer compound of Group C-1 or Group C-2characterized in that the temperature-responsiveness thereof can becontrolled by changing the composition or functional groups of themonomers constituting said polymer compound, the molecular weight ofsaid polymer compound or the concentration of said polymer compound in asolution.

The present invention furthermore provides an adsorption and separationmaterial characterized by containing the temperature-responsive polymercompound represented by the formula of Group C-1 or Group C-2. Thismaterial can be used in the kind of separation methods described abovein which the stationary phase contains a polymer compound from group C-1or C-2, possibly replacing the Group A or Group B polymer compound.

The present invention provides a polymer compound containing a repeatingunit represented by the formula of Group D or a copolymer or gelstructure containing this unit structure and showingtemperature-responsiveness in a solution, a process for producing thesame, and a material for separating, adsorbing and releasing a substancewith the use of the same. In addition this aspect of the inventionprovides a separation method in analogy with those described above butwith the stationary phase comprising a polymer compound selected fromgroup D, possibly together with group A, B, C, and E polymers.

The present invention furthermore provides materials for separating,adsorbing and releasing substances characterized by containing theabove-mentioned temperature-responsive polymer compound represented bythe formula of Group E-1, Group E-2, Group E-3, Group E-4 or Group E-5.

In addition, the present invention provides a method for separatingsubstances characterized by providing a chromatographic packing to whichat least one of the polymer compounds of the formula of Group E-1, GroupE-2, Group E-3, Group E-4 or Group E-5 is attached and then;

-   -   (i) binding/adsorbing a substances to the stationary phase;    -   (ii) changing the hydrophobic-hydrophilic balance and/or the        hydrogen-bonding properties of the stationary phase by changing        the temperature of the phase, preferably by external means and        preferably in one or more steps;    -   (iii) passing a mobile phase, preferably of constant composition        and preferably in liquid form, through the packing;        steps (ii) and (iii) effecting release and separation of the        substance from the packing/stationary phase.

The present invention further provides a process for producing themonomer represented by the formula of Group E-1 which comprises reactingacrylic acid chloride, methacrylic acid chloride, anhydrous acrylic acidor anhydrous methacrylic acid with a cyclic secondary amine compoundhaving an amide group.

The present invention further provides a process for producing themonomer represented by the formula of Group E-3 which comprises reactingacrylic acid chloride, methacrylic acid chloride, anhydrous acrylic acidor anhydrous methacrylic acid with a secondary amine compound having atleast one acyl bond.

Moreover, the present invention provides a process for producing polymercompounds which comprises adding the monomers represented by theformulae of Group E-1, Group E-2, Group E-3 and Group E-4 optionallytogether with a polymerization initiator to a polymerization solvent andthen polymerizing the same under light-irradiation or at such atemperature as to induce the formation of a radical from thepolymerization initiator.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 provides a graph showing a relationship between the permeabilityand temperature of an aqueous solution ofpoly-methacryloyl-acetylaminoethyl-ester, obtained in Example A1.

FIG. 2 provides a spectrogram showing the results of mass spectrometricanalysis of the purified product obtained in Example B1.

FIG. 3 provides a spectrogram showing the results of ¹H-NMR analysis ofthe purified product obtained in Example B1.

FIG. 4 provides a graph showing a relationship between the permeabilityand temperature of the polymer obtained in Example B5.

FIG. 5 provides a graph showing a relationship between the permeabilityand temperature of the polymer obtained in Example B6.

FIG. 6 provides a graph showing a relationship between the permeabilityand temperature of the polymer obtained in Example B7.

FIG. 7 provides a graph which shows the expression of thetemperature-responsiveness bypoly(acrylamide-co-3-acrylamideacetanilide) in an aqueous solutionobatined in Example C2.

FIG. 8 provides a graph which shows the expression of thetemperature-responsiveness bypoly(glycosyloxyethyl-methacrylate-co-4-acrylamidebenzamide) in anaqueous solution obtained in Example C10.

FIG. 9 provides a graph which shows Vant 'Hoff plot of cortisone acetateby silica gel carryingpoly(glycosyloxy-ethylmethacrylate-co-4-acrylamidebenzamide) fixedthereto obtained in Example C10.

FIG. 10 provides a graph showing the expression of thetemperature-responsiveness of poly(5-hydroxypentyl-acrylamide) in anaqueous solution obtained in Example D1.

FIG. 11 provides a graph showing the expression of thetemperature-responsiveness of poly(trans-hydroxycyclo-hexylacrylamide)in an aqueous solution obtained in Example D2.

FIG. 12 provides a graph showing the expression of thetemperature-responsiveness of poly(6-hydroxyhexylacrylamide) in aqueoussolutions obtained in Example D4.

FIG. 13 provides a graph showing the expression of thetemperature-responsiveness of poly(4-piperidinecarboxamide) in ammoniumsulfate solutions obtained in Example E3.

FIG. 14 provides a graph showing the expression of thetemperature-responsiveness of poly(4-piperidinecarboxamide) in anammonium sulfate solution under elevating and lowering temperatureobtained in Example E3.

FIG. 15 provides a graph showing that the temperature-responsiveness ofpoly(acrylamide-co-3-acrylamide acetanilide) can be controlled dependingon salt concentration obtained in Example E4.

BEST MODE FOR CARRYING OUT THE INVENTION

The polymer compound according to the present invention has thefollowing structure.

1. Compounds represented by the formula of Group A.

wherein R represents a linear or branched divalent aliphatic hydrocarbongroup having 1 to 8 carbon atoms, a divalent alicyclic hydrocarbon grouphaving 3 to 8 carbon atoms, or a divalent aromatic hydrocarbon grouphaving 6 to 14 carbon atoms; R′ represents a linear or branchedaliphatic hydrocarbon group having 1 to 8 carbon atoms, a linear orbranched aliphatic hydrocarbon group having one or more hydroxyl groupsand 1 to 8 carbon atoms, a linear or branched aliphatic hydrocarbongroup having one or more acid amide bonds and/or ester bonds and 2 to 9carbon atoms, or a linear or branched aliphatic hydrocarbon group havingone or more acid amide bonds and/or ester bonds, one or more hydroxylgroups and 3 to 9 carbon atoms; and n is an integer of 2 or above.

The term a “linear or branched aliphatic hydrocarbon group having 1 to 8carbon atoms” as used in the formula of Group A of the present inventionmeans a linear or branched alkyl group having 1 to 8 carbon atoms, alinear or branched alkenyl group having 2 to 8 carbon atoms or a linearor branched alkynyl group having 2 to 8 carbon atoms. The alkenyl grouphas one or more double bonds, while the alkynyl group has one or moretriple bonds. Preferable examples of the alkyl group include methyl,ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl and t-butyl groups.Preferable examples of the alkenyl group include vinyl, 1-propenyl,2-propenyl(allyl), 1-butenyl, 2-butenyl and 3-butenyl groups. Preferableexamples of the alkynyl groups include ethynyl, 1-propynyl, 2-propynyl(propargyl), 1-butynyl and 2-butynyl groups.

The term a “linear or branched divalent aliphatic hydrocarbon grouphaving 1 to 8 carbon atoms” as used in the formula of Group A of thepresent invention means a linear or branched divalent alkyl group having1 to 8 carbon atoms; a linear or branched divalent alkenyl group having2 to 8 carbon atoms or a linear or branched divalent alkynyl grouphaving 2 to 8 carbon atoms. The alkenyl group has one or more doublebonds, while the alkynyl group has one or more triple bonds. Preferableexamples of the linear or branched divalent alkyl group having 1 to 8carbon atoms include methylene, ethylene, ethylidene, trimethylene,propylene (1,2-propanediyl), isopropylidene, tetramethylene,ethylethylene, pentamethylene and hexamethylene groups. Preferableexamples of the linear or branched divalent alkenyl group having 2 to 8carbon atoms include vinylene, vinylidene, propenylene, 1-butenylene,2-butenylene, 1-pentenylene, 2-pentenylene, 1-hexenylene, 2-hexenyleneand 3-hexenylene groups. Preferable examples of the linear or brancheddivalent alkynyl group having 2 to 8 carbon atoms include ethynylene,propynylene, 1-butynylene and 2-butynylene groups.

The term an “alicyclic hydrocarbon group having 3 to 8 carbon atoms” asused in the formula of Group A of the present invention means acycloalkyl group having 3 to 8 carbon atoms or a cycloalkenyl grouphaving 3 to 8 carbon atoms. The cycloalkenyl group has one or moredouble bonds. Preferable examples of the cycloalkyl group includecyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl groups. Preferableexamples of the cycloalkenyl group include 1-cyclopropen-1-yl,2-cyclopropen-1-yl, 1-cyclobuten-1-yl, 2-cyclobuten-1-yl,1-cyclopenten-1-yl, 2-cyclopenten-1-yl, 3-cyclopenten-1-yl,1-cyclohexen-1-yl, 2-cyclohexn-1-yl and 3-cyclohexen-1-yl groups.

The term a “divalent alicyclic hydrocarbon group having 3 to 8 carbonatoms” as used in the formula of Group A of the present invention meansa divalent cycloalkyl group having 3 to 8 carbon atoms or a divalentcycloalkenyl group having 3 to 8 carbon atoms. Preferable examples ofthe divalent cycloalkyl group having 3 to 8 carbon atoms include1,2-cyclopropylene, 1,2-cyclobutylene, 1,3-cyclobutylene,1,2-cyclopentylene, 1,3-cyclopentylene, 1,2-cyclohexylene,1,3-cyclohexylene, 1,4-cyclohexylene, 1,2-cyclooctylene,1,3-cyclooctylene, 1,4-cyclooctylene and 1,5-cyclooctylene groups.Preferable examples of the divalent cycloalkenyl group having 3 to 8carbon atoms include 1-cyclopropen-1,2-enylene,1-cyclobuten-1,2-enylene, 1-cyclobuten-3,4-ylene,1-cyclohexen-1,2-enylene, 3-cyclohexen-1,2-ylene, 4-cyclohexen-1,2-yleneand 2,5-cyclohexadien-1,4-ylene.

The term an “aromatic hydrocarbon group having 6 to 14 carbon atoms” asused in the formula of Group A of the present invention means an aryl oraralkyl group having 6 to 14 carbon atoms. Preferable examples of thearomatic hydrocarbon group include phenyl, benzyl, phenethyl,1-naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl, 9-anthryl, 1-phenanthryl,2-phenanthryl, 3-phenanthryl, 4-phenanthryl and 9-phenanthryl groups.

The term a “divalent aromatic hydrocarbon group having 6 to 14 carbonatoms” as used in the formula of Group A of the present invention meansa divalent aryl group or a divalent aralkyl group having 6 to 14 carbonatoms. Preferable examples of the divalent aromatic hydrocarbon grouphaving 6 to 14 carbon atoms include o-phenylene, m-phenylene,p-phenylene, -o-φ-CH₂—, -m-φ-CH₂— and -p-φ-CH₂— groups wherein φrepresents a benzene ring.

The term a “linear or branched aliphatic hydrocarbon group having one ormore hydroxyl groups and 1 to 8 carbon atoms” as used in the formula ofGroup A of the present invention means an above-mentioned linear orbranched aliphatic hydrocarbon group having 1 to 8 carbon atoms withsubstitution by one or more hydroxyl groups at arbitrary carbon atom(s).When it has two hydroxyl groups, these two hydroxyl groups may beattached to a carbon atom. Preferable examples thereof includehydroxymethyl, 2-hydroxyethyl, 1-hydroxyethyl, 1-hydroxy-n-propyl,2-hydroxy-n-propyl, 3-hydroxy-n-propyl, 1-hydroxy-1-methylethyl,2-hydroxy-1-methylethyl, 1-hydroxy-n-butyl, 2-hydroxy-n-butyl,3-hydroxy-n-butyl, 4-hydroxy-n-butyl, 1-hydroxy-1-methyl-n-propyl,2-hdyroxy-1-methyl-n-propyl, 3-hydroxy-1-methyl-n-propyl,1-hydroxy-2-methyl-n-propyl, 2-hdyroxy-2-methyl-n-propyl,3-hydroxy-2-methyl-n-propyl, 1-hydroxymethyl-1-methylethyl,2-hydroxy-1,1-dimethylethyl, 1-hydroxyvinyl, 2-hydroxyvinyl,1-hydroxyallyl, 2-hydroxyallyl, 3-hydroxyallyl, 2-hydroxy-1-methylvinyland 1-hydroxymethylvinyl groups.

The term a “linear or branched aliphatic hydrocarbon group having one ormore acid amide bonds and/or ester bonds and 2 to 9 carbon atoms” asused in the formula of Group A of the present invention means a linearor branched alkyl, alkenyl or alkynyl group with substitution by a grouphaving one or more acid amide bonds and/or ester bonds at arbitrarycarbon atom(s) which has 2 to 9 carbon atoms in total. Preferableexamples thereof include acetyloxymethyl, 2-acetyloxy-ethyl,3-acetyloxy-n-propyl, 1-acetyloxy-1,1-dimethyl-methyl,4-acetyloxy-n-butyl, 2-acetyloxy-1,1-dimethyl-ethyl,3-acetyloxy-1-methyl-n-propyl, 2-acetyloxy-2,2-dimethyl-ethyl,propionyloxymethyl, 2-propionyloxy-ethyl, 3-propionyloxy-n-propyl,1-propionyloxy-1,1-dimethyl-methyl, acetamidomethyl, 2-acetamido-ethyl,3-acetamido-n-propyl, 1-acetamido-1,1-dimethyl-ethyl,propionylaminomethyl, 2-propionylamino-ethyl, 3-propionylamino-n-propyland 1-propionylamino-1,1-dimethyl-ethyl groups.

The term a “linear or branched aliphatic hydrocarbon group having one ormore acid amide bonds and/or ester bonds, one or more hydroxyl groupsand 3 to 9 carbon atoms” as used in the formula of Group A of thepresent invention means a linear or branched alkyl, alkenyl or alkynylgroup with substitution by a group having one or more acid amide bondsand/or ester bonds and one or more hydroxyl groups at arbitrary carbonatom(s) which has 3 to 9 carbon atoms in total. Preferable examplesthereof include (1-hydroxypropionate)methyl,(1-hydroxypropionate)-ethyl, (1-hydroxypripionamino)methyl and(1-hydroxypropion-amino)ethyl groups.

In the polymer compound represented by the above formula in the formulaof Group A of the present invention, n is 2 or above. The value n may becontrolled appropriately depending on the substance to be separated,etc. It is desirable that n is 5 or above.

The polymer compound represented by the formula of Group A according tothe present invention can be obtained by one of the following methods.

(1) A monomer having a primary amino group (2-aminoethyl methacrylate,2-aminoethyl methacrylate hydrochloride, etc.) is reacted with an acidanhydride (acetic anhydride, propionic anhydride, etc.) or lactone(propyl lactone, butyl lactone, etc.). The thus obtained product ispurified by using a column and then polymerized in an appropriatesolvent (methanol, ethanol, dimethyl sulfoxide, etc.). In this case, thesubstituent R in the polymer compound represented by the above formulacan be controlled in size depending on the type of the monomer, whilethe substituent R′ therein can be also controlled in size depending onthe acid anhydride or lactone employed.

(2) A monomer having a hydroxyl group (2-hydroxethyl methacrylate, etc.)is reacted with an acid chloride (acetyl chloride, propionyl chloride,etc.). The thus obtained product was purified and then polymerized in anappropriate solvent (methanol, ethanol, dimethyl sulfoxide, etc.). Inthis case, the substituent R in the polymer compound represented by theabove formula can be controlled in size depending on the type of themonomer, while the substituent R′ therein can be also controlled in sizedepending on the acid chloride.

(3) An alkylamino alcohol (3-amino propanol, amino ethanol, etc.) isreacted with an acid anhydride (acetic anhydride, propionic anhydride,etc.). Then the thus obtained product is further reacted with acrylicacid chloride or methacrylic acid chloride. The resulting product ispurified and then polymerized in an appropriate solvent (methanol,ethanol, dimethyl sulfoxide, etc.). In this case, the substituents R andR′ in the polymer compound represented by the above formula can becontrolled in size depending respectively on the alkylamino alcohol andthe acid anhydride.

(4) Poly-2-aminoethyl methacrylate or its hydrochloride is synthesizedand then reacted with an acid anhydride (acetic anhydride, propionicanhydride, etc.) or lactone (propyl lactone, butyl lactone, etc.) in asolvent containing triethylamine (TEA). In this case, the substituent R′can be controlled in size depending on the lactone or acid anhydride.

The term an “acid anhydride” means a carboxylic acid anhydrideexemplified by acetic anhydride, propionic anhydride, butyric anhydride,isobutyric anhydride, valeric anhydride, isovaleric anhydride, capronicanhydride, maleic anhydride, etc.

The lactone is exemplified by β-propiolactone, γ-butyl lactone,γ-valerolactone, δ-valerolactone, γ-hexanolactone, δ-hexanolactone,ε-caprolactone, α-amino-γ-butyrolactone having an optically activegroup, etc.

The alkyl acid chloride is exemplified by acetyl chloride, propionylchloride, butyrl chloride, valeroyl chloride, etc.

The reaction between the aminoalkyl(meth)acrylate(hydrochloride)[wherein the term “aminoalkyl(meth)acrylate hydrochloride” means onemember selected from among aminoalkyl acrylate, aminoalkyl acrylatehydrochloride, aminoalkyl methacrylate and aminoalkyl methacrylatehydrochloride] and the acid anhydride is performed by adding a basiccompound (for example, TEA) into a solvent (for example, an alcohol) andthen slowly dropping one of the reactants thereinto. This reaction iscarried out under ice-cooling and it is desirable to use methanol as thesolvent.

After the completion of the reaction, the solvent is eliminated with anevaporator and the precipitate is taken up by filtration. Next, thetarget product is separated by column chromatography and purified byrecrystallization.

The monomer thus purified is polymerized in an aqueous solution ororganic solvent containing a polymerization initiator. After thecompletion of the polymerization reaction, reprecipitation is carriedout in a solvent (for example, alcohol, acetone, ether, or a mixturethereof) to give the aimed polymer.

The cloud point of the obtained polymer can be arbitrarily controlled byvarying the carbon atom number in the polymer side chain, the molecularweight, or the salt concentration. For example, the cloud point can becontrolled by varying the concentration of the polymerization initiatoror the monomer or by using a chain transfer agent such as3-mercaptopropionic acid.

Alternatively, polymer materials having various cloud points can besynthesized by copolymerizing with other alkyl acrylamides or alkylmethacrylamides (N-isopropyl acrylamide, N-isopropyl methacrylamide,N-n-propyl acrylamide, N-n-propyl methacrylamide, etc.), or alkylacrylates or alkyl methacrylates (butyl acrylate, butyl methacrylate,hydroxyethyl acrylate, hydroxyethyl methacrylate, glycidyl acrylate,glycidyl methacrylate, etc.).

It is also possible to synthesize a polymer the cloud point of which canbe controlled depending on the pH environment by copolymerizing with ananionic monomer (acrylic acid, methacrylic acid, etc.) or a cationicmonomer (acryloxy-ethyltriethylammonium,methacryloxyethyltriethylammonium, etc.).

It is considered that the phenomenon of the cloud point of theheat-responsive polymer is induced by the breakage or formation of thehydration water in the side chain or the intramolecular orintermolecular interaction among the polymer chains.

2. Compounds represented by the formula of Group B.

wherein R represents a linear or branched divalent aliphatic hydrocarbongroup having 1 to 8 carbon atoms, a divalent alicyclic hydrocarbon grouphaving 3 to 8 carbon atoms, or a divalent aromatic hydrocarbon grouphaving 6 to 14 carbon atoms; R′ represents a linear or branchedaliphatic hydrocarbon group having 1 to 8 carbon atoms, a linear orbranched aliphatic hydrocarbon group having one or more hydroxyl groupsand 1 to 8 carbon atoms, a linear or branched aliphatic hydrocarbongroup having one or more acid amide bonds and/or ester bonds and 2 to 9carbon atoms, or a linear or branched aliphatic hydrocarbon group havingone or more acid amide bonds and/or ester bonds, one or more hydroxylgroups and 3 to 9 carbon atoms; and n is an integer of 2 or above.

The term a “linear or branched aliphatic hydrocarbon group having 1 to 8carbon atoms” as used in the formula of Group B of the present inventionmeans a linear or branched alkyl group having 1 to 8 carbon atoms, alinear or branched alkenyl group having 2 to 8 carbon atoms or a linearor branched alkynyl group having 2 to 8 carbon atoms. The alkenyl grouphas one or more double bonds, while the alkynyl group has one or moretriple bonds. Preferable examples of the alkyl group include methyl,ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl and t-butyl groups.Preferable examples of the alkenyl group include vinyl, 1-propenyl,2-propenyl(allyl), 1-butenyl, 2-butenyl and 3-butenyl groups. Preferableexamples of the alkynyl groups include ethynyl, 1-propynyl, 2-propynyl(propargyl), 1-butynyl and 2-butynyl groups.

The term a “linear or branched divalent aliphatic hydrocarbon grouphaving 1 to 8 carbon atoms” as used in the formula of Group B of thepresent invention means a linear or branched divalent alkyl group having1 to 8 carbon atoms, a linear or branched divalent alkenyl group having2 to 8 carbon atoms or a linear or branched divalent alkynyl grouphaving 2 to 8 carbon atoms. The alkenyl group has one or more doublebonds, while the alkynyl group has one or more triple bonds. Preferableexamples of the linear or branched divalent alkyl group having 1 to 8carbon atoms include methylene, ethylene, ethylidene, trimethylene,propylene (1,2-propanediyl), isopropylidene, tetramethylene,ethylethylene, pentamethylene, hexamethylene, heptamethylene andoctamethylene groups. Preferable examples of the linear or brancheddivalent alkenyl group having 2 to 8 carbon atoms include vinylene,vinylidene, propenylene, 1-butenylene, 2-butenylene, 1-pentenylene,2-pentenylene, 1-hexenylene, 2-hexenylene and 3-hexenylene groups.Preferable examples of the linear or branched divalent alkynyl grouphaving 2 to 8 carbon atoms include ethynylene, propynylene, 1-butynyleneand 2-butynylene groups.

The term an “alicyclic hydrocarbon group having 3 to 8 carbon atoms” asused in the formula of Group B of the present invention means acycloalkyl group having 3 to 8 carbon atoms or a cycloalkenyl grouphaving 3 to 8 carbon atoms. The cycloalkenyl group has one or moredouble bonds. Preferable examples of the cycloalkyl group includecyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl groups. Preferableexamples of the cycloalkenyl group include 1-cyclopropen-1-yl,2-cyclopropen-1-yl, 1-cyclobuten-1-yl, 2-cyclobuten-1-yl,1-cyclopenten-1-yl, 2-cyclopenten-1-yl, 3-cyclopenten-1-yl,1-cyclohexen-1-yl, 2-cyclohexn-1-yl and 3-cyclohexen-1-yl groups.

The term a “divalent alicyclic hydrocarbon group having 3 to 8 carbonatoms” as used in the formula of Group B of the present invention meansa divalent cycloalkyl group having 3 to 8 carbon atoms or a divalentcycloalkenyl group having 3 to 8 carbon atoms. Preferable examples ofthe divalent cycloalkyl group having 3 to 8 carbon atoms include1,2-cyclopropylene, 1,2-cyclobutylene, 1,3-cyclobutylene,1,2-cyclopentylene, 1,3-cyclopentylene, 1,2-cyclohexylene,1,3-cyclohexylene, 1,4-cyclohexylene, 1,2-cyclooctylene,1,3-cyclooctylene, 1,4-cyclooctylene and 1,5-cyclooctylene groups.Preferable examples of the divalent cycloalkenyl group having 3 to 8carbon atoms include 1-cyclopropen-1,2-enylene,1-cyclobuten-1,2-enylene, 1-cyclobuten-3,4-ylene,1-cyclohexen-1,2-enylene, 3-cyclohexen-1,2-ylene, 4-cyclohexen-1,2-yleneand 2,5-cyclohexadien-1,4-ylene.

The term an “aromatic hydrocarbon group having 6 to 14 carbon atoms” asused in the formula of Group B of the present invention means an aryl oraralkyl group having 6 to 14 carbon atoms. Preferable examples of thearomatic hydrocarbon group include phenyl, benzyl, phenethyl,1-naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl, 9-anthryl, 1-phenanthryl,2-phenanthryl, 3-phenanthryl, 4-phenanthryl and 9-phenanthryl groups.

The term a “divalent aromatic hydrocarbon group having 6 to 14 carbonatoms” as used in the formula of Group B of the present invention meansa divalent aryl group or a divalent aralkyl group having 6 to 14 carbonatoms. Preferable examples of the divalent aromatic hydrocarbon grouphaving 6 to 14 carbon atoms include o-phenylene, m-phenylene,p-phenylene, -o-φ-CH₂—, -m-φ-CH₂— and -p-φ-CH₂— groups wherein 4represents a benzene ring.

The term a “linear or branched aliphatic hydrocarbon group having one ormore hydroxyl groups and 1 to 8 carbon atoms” as used in the formula ofGroup B of the present invention means an above-mentioned linear orbranched aliphatic hydrocarbon group having 1 to 8 carbon atoms withsubstitution by one or more hydroxyl groups at arbitrary carbon atom(s).When it has two hydroxyl groups, these two hydroxyl groups may beattached to a carbon atom. Preferable examples thereof includehydroxymethyl, 2-hydroxyethyl, 1-hydroxyethyl, 1-hydroxy-n-propyl,2-hydroxy-n-propyl, 3-hydroxy-n-propyl, 1-hydroxy-1-methylethyl,2-hydroxy-1-methylethyl, 1-hydroxy-n-butyl, 2-hydroxy-n-butyl,3-hydroxy-n-butyl, 4-hydroxy-n-butyl, 1-hydroxy-1-methyl-n-propyl,2-hdyroxy-1-methyl-n-propyl, 3-hydroxy-1-methyl-n-propyl,1-hydroxy-2-methyl-n-propyl, 2-hdyroxy-2-methyl-n-propyl,3-hydroxy-2-methyl-n-propyl, 1-hydroxymethyl-1-methylethyl,2-hydroxy-1,1-dimethylethyl, 1-hydroxyvinyl, 2-hydroxyvinyl,1-hydroxyallyl, 2-hydroxyallyl, 3-hydroxyallyl, 2-hydroxy-1-methylvinyland 1-hydroxymethylvinyl groups.

The term a “linear or branched aliphatic hydrocarbon group having one ormore acid amide bonds and/or ester bonds and 2 to 9 carbon atoms” asused in the formula of Group B of the present invention means a linearor branched alkyl, alkenyl or alkynyl group with substitution by a grouphaving one or more acid amide bonds and/or ester bonds at arbitrarycarbon atom(s) which has 2 to 9 carbon atoms in total. Preferableexamples thereof include acetyloxymethyl, 2-acetyloxy-ethyl,3-acetyloxy-n-propyl, 1-acetyloxy-1,1-dimethyl-methyl,4-acetyloxy-n-butyl, 2-acetyloxy-1,1-dimethyl-ethyl,3-acetyloxy-1-methyl-n-propyl, 2-acetyloxy-2,2-dimethyl-ethyl,propionyloxymethyl, 2-propionyloxy-ethyl, 3-propionyloxy-n-propyl,1-propionyloxy-1,1-dimethyl-methyl, acetamidomethyl, 2-acetamido-ethyl,3-acetamido-n-propyl, 1-acetamido-1,1-dimethyl-ethyl,propionylaminomethyl, 2-propionylamino-ethyl, 3-propionylamino-n-propyland 1-propionylamino-1,1-dimethyl-methyl groups.

The term a “linear or branched aliphatic hydrocarbon group having one ormore acid amide bonds and/or ester bonds, one or more hydroxyl groupsand 3 to 9 carbon atoms” as used in the formula of Group B of thepresent invention means a linear or branched alkyl, alkenyl or alkynylgroup with substitution by a group having one or more acid amide bondsand/or ester bonds and one or more hydroxyl groups at arbitrary carbonatom(s) which has 3 to 9 carbon atoms in total. Preferable examplesthereof include (1-hydroxypropionate)methyl,(1-hydroxypropionate)-ethyl, (1-hydroxypripionamino)methyl and(1-hydroxypropion-amino)ethyl groups.

In the polymer compound represented by the formula of Group B in thepresent invention, n is 2 or above. The value n may be controlledappropriately depending on the substance to be separated, etc. It isdesirable that n is 5 or above.

The polymer compound represented by the formula of Group B according tothe present invention can be obtained by one of the following methods.

(1) A compound selected from among aminoalkyl acylamide, aminoalkylmethacrylamide, aminoalkyl acrylamide hydrochloride and aminoalkylmethacrylamide hydrochloride is reacted with an acid anhydride (aceticanhydride, propionic anhydride, etc.) or lactone (propyl lactone, butyllactone, etc.). The thus obtained product is purified by using a columnand then polymerized in an appropriate solvent (methanol, ethanol,dimethyl sulfoxide, etc.). In this case, the substituent R in thepolymer compound represented by the above formula can be controlled insize depending on the size of the alkyl group carried by the monomerhaving an amino group, while the substituent R′ therein can be alsocontrolled in size depending on the acid anhydride or lactone employed.

(2) An alkyl diamine (ethylene diamine, trimethylene diamine,tetramethylene diamine, pentamethylene diamine, hexamethylene diamine,etc.) is reacted with an acid anhydride (acetic anhydride, propionicanhydride, etc.), an alkyl acid chloride (acetyl chloride, propionylchloride, etc.) or di-t-butyl dicarbonate. Alternatively, a compoundhaving an amino group and an amide bond in its molecule is reacted withacryloyl chloride or methacryloyl chloride. Then the thus obtainedproduct is purified followed by polymerization in a solvent (methanol,ethanol, dimethyl sulfoxide, etc.). In this case, the substituent R inthe polymer compound represented by the above formula can be controlledin size depending on the size of the alkylene in the alkyl diamine,while the substituent R′ therein can be also controlled in sizedepending on the acid chloride.

The term an “acid anhydride” means a carboxylic acid anhydrideexemplified by acetic anhydride, propionic anhydride, butyric anhydride,isobutyric anhydride, valeric anhydride, isovaleric anhydride, capronicanhydride, maleic anhydride, etc.

The lactone is exemplified by β-propiolactone, γ-butyl lactone,γ-valerolactone, δ-valerolactone, γ-hexanolactone, δ-hexanolactone,ε-caprolactone, α-amino-γ-butyrolactone having an optically activegroup, etc.

The reaction between the aminoalkyl acrylate, aminoalkyl acrylatehydrochloride, aminoalkyl methacrylate and aminoalkyl methacrylatehydrochloride and the acid anhydride is performed by adding a basiccompound (for example, TEA) into a solvent (for example, an alcohol) andthen slowly dropping one of the reactants thereinto. This reaction iscarried out under ice-cooling and it is desirable to use methanol as thesolvent.

After the completion of the reaction, the solvent is eliminated with anevaporator and the precipitate is taken up by filtration. Next, thetarget product is separated by column chromatography and purified byrecrystallization.

The monomer thus purified is polymerized in an aqueous solution ororganic solvent containing a polymerization initiator. After thecompletion of the polymerization reaction, reprecipitation is carriedout in a solvent (for example, alcohol, acetone, ether, or a mixturethereof) to give the aimed polymer.

In the present invention, a similar compound can be obtained by reactingan alkyl diamines (1,3-propyldiamine, ethylene diamine,1,6-hexamethylene diamine, 1,2-propane diamine, etc.) or a diamine(spermine, spermidine, etc.) with the equimolar amount of an acidanhydride or an alkyl acid chloride, further reacting the resultantproduct with acryloyl chloride or methacryloyl chloride and polymerizingthe thus obtained monomer.

The cloud point of the obtained polymer can be arbitrarily controlled byvarying the carbon atom number in the polymer side chain, the molecularweight, or the salt concentration. For example, the cloud point isliable to be lowered with an increase in the salt concentration of theaqueous solution.

Alternatively, polymer materials having various cloud points can besynthesized by copolymerizing with other alkyl acrylamides or alkylmethacrylamides (N-isopropyl acrylamide, N-isopropyl methacrylamide,N-n-propyl acrylamide, N-n-propyl methacrylamide, etc.), or alkylacrylates or alkyl methacrylates (butyl acrylate, butyl methacrylate,hydroxyethyl acrylate, hydroxyethyl methacrylate, glycidyl acrylate,glycidyl methacrylate, etc.). A copolymer with n-butyl acrylamide showsa lowered cloud point, while one with acrylamide shows an elevated cloudpoint.

It is also possible to synthesize a polymer the cloud point of which canbe controlled depending on the pH environment by copolymerizing with ananionic monomer (acrylic acid, methacrylic acid, etc.) or a cationicmonomer (acryloxy-ethyltriethylammonium,methacryloxyethyltriethylammonium, etc.).

It is considered that the phenomenon of the cloud point of theheat-responsive polymer is induced by the breakage or formation of thehydration water in the side chain or the intramolecular orintermolecular interaction among the polymer chains.

3. Compounds represented by the formulae of Group C-1 and Group C-2.

In the above formula of Group C-1, n is from 0.005 to 0.995 (inclusive),while j is from 0 to 0.5 (inclusive). R¹, R², R³ and R⁴ are the same ordifferent and each represents a hydrogen atom or a methyl group. X¹, X²,X² and X⁴ are the same or different and each represents acid amidesgroup, an ester group or an ether group. Y¹ represents a linear orbranched divalent aliphatic hydrocarbon group having 1 to 8 carbonatoms, a divalent alicyclic hydrocarbon group having 3 to 8 carbon atomsor a divalent aromatic hydrocarbon group having 6 to 14 carbon atoms. Y²represents a linear or branched divalent aliphatic hydrocarbon grouphaving 1 to 8 carbon atoms, a divalent alicyclic hydrocarbon grouphaving 3 to 8 carbon atoms, a linear or branched divalent aliphatichydrocarbon group having 1 to 8 carbon atoms and one or more ethergroups or a linear or branched divalent aliphatic hydrocarbon grouphaving 1 to 8 carbon atoms and one or more hydroxyl groups. Z¹, Z², Z³,Z⁵ and Z⁶ are the same or different and each represents a hydrogen atom,a linear or branched aliphatic hydrocarbon group having 1 to 8 carbonatoms, a linear or branched aliphatic hydrocarbon group having 1 to 8carbon atoms and one or more hydroxyl groups, a linear or branchedalicyclic hydrocarbon group having 1 to 8 carbon atoms and one or morehydroxyl group, a linear or branched aliphatic hydrocarbon group having1 to 8 carbon atoms and one or more ether groups, a linear or branchedalicyclic hydrocarbon group having 1 to 8 carbon atoms and one or moreether groups, a glycoside having 3 to 12 carbon atoms or a glycosidehaving 3 to 12 carbon atoms and carrying a linear or branched aliphatichydrocarbon group having 1 to 8 carbon atoms, provided that Z¹, Z³, Z⁵and Z⁶ are functional groups bonded respectively to X¹, X², X³ and X⁴when they are tertiary amide groups and Z⁵ may be bonded to Z⁶. Z⁴represents a hydrogen atom, a hydroxyl group, an amide group, a nitrylgroup, a linear or branched aliphatic hydrocarbon group having 1 to 8carbon atoms, a linear or branched aliphatic hydrocarbon group having 1to 8 carbon atoms and one or more amide groups, a linear or branchedaliphatic hydrocarbon group having 1 to 8 carbon atoms and one or morecarbonyl groups, a linear or branched aliphatic hydrocarbon group having1 to 8 carbon atoms and one or more nitryl groups, or a linear orbranched aliphatic hydrocarbon group having 1 to 8 carbon atoms and oneor more hydroxyl groups.

In the above formula, R⁵ represents a hydrogen atom or a methyl group.X⁵ represents an acid amide group, an ester group or an ether group. Y³represents a linear or branched divalent aliphatic hydrocarbon grouphaving 1 to 8 carbon atoms or a divalent alicyclic hydrocarbon grouphaving 3 to 8 carbon atoms. Y⁴ represents a linear or branched aliphatichydrocarbon group having 1 to 8 carbon atoms or a linear or branchedaliphatic hydrocarbon group having 1 to 8 carbon atoms and one or morehydroxyl groups.

The term a “linear or branched aliphatic hydrocarbon group having 1 to 8carbon atoms” as used in the formula of Group C-1 herein means a linearor branched alkyl group having 1 to 8 carbon atoms, a linear or branchedalkenyl group having 1 to 8 carbon atoms or a linear or branched alkynylgroup having 1 to 8 carbon atoms. The alkenyl group may have one or moredouble bonds while the alkynyl group may have one or more triple bonds.Preferable examples of the alkyl group include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, n-hexyl,n-heptyl and n-octyl groups. Preferable examples of the alkenyl groupinclude vinyl, propenyl (1-propenyl), allyl, butenyl (wherein the doublebond may be at an arbitrary site), pentenyl (wherein the double bond maybe at an arbitrary site), hexenyl (wherein the double bond may be at anarbitrary site), heptenyl (wherein the double bond may be at anarbitrary site) and octenyl (wherein the double bond may be at anarbitrary site) groups. Preferable examples of the alkynyl group includeethynyl, propargyl, butynyl (wherein the triple bond may be at anarbitrary site), pentynyl (wherein the triple bond may be at anarbitrary site), hexynyl (wherein the triple bond may be at an arbitrarysite), heptynyl (wherein the triple bond may be at an arbitrary site)and octynyl (wherein the triple bond may be at an arbitrary site)groups.

The term a “linear or branched divalent aliphatic hydrocarbon grouphaving 1 to 8 carbon atoms” as used in the formulae of Group C-1 andGroup C-2 herein means a linear or branched alkylene group having 1 to 8carbon atoms, a linear or branched alkenylene group having 1 to 8 carbonatoms or a linear or branched alkynylene group having 1 to 8 carbonatoms. The alkenylene group may have one or more double bonds while thealkynylene group may have one or more triple bonds. Preferable examplesof the alkylene group include methylene, ethylene, propylene, butylene,pentylene, hexylene, heptylene and octylene groups. Preferable examplesof the alkenylene group include vinylene, propenylene (wherein thedouble bond may be at an arbitrary site), butenylene (wherein the doublebond may be at an arbitrary site), pentenylene (wherein the double bondmay be at an arbitrary site), hexenylene (wherein the double bond may beat an arbitrary site), heptenylene (wherein the double bond may be at anarbitrary site) and octenylene (wherein the double bond may be at anarbitrary site) groups. Preferable examples of the alkynylene groupinclude ethynylene, propynylene (wherein the triple bond may be at anarbitrary site), butynylene (wherein the triple bond may be at anarbitrary site), pentynylene (wherein the triple bond may be at anarbitrary site), hexynylene (wherein the triple bond may be at anarbitrary site), heptynylene (wherein the triple bond may be at anarbitrary site) and octynylene (wherein the triple bond may be at anarbitrary site) groups.

The term a “divalent alicyclic hydrocarbon group having 3 to 8 carbonatoms” as used in the formulae of Group C-1 and Group C-2 herein means acycloalkylene group having 3 to 8 carbon atoms or a cycloalkenylenegroup having 3 to 8 carbon atoms. The cycloalkenylene group may have oneor more double bonds. Preferable examples of the cycloalkylene groupinclude cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene,cycloheptylene and cyclooctylene groups. Preferable examples of thecycloalkenylene group include cyclopropenylene (wherein the double bondmay be at an arbitrary site), cyclobutenylene (wherein the double bondmay be at an arbitrary site), cyclopentenylene (wherein the double bondmay be at an arbitrary site), cyclohexenylene (wherein the double bondmay be at an arbitrary site), cycloheptenylene (wherein the double bondmay be at an arbitrary site) and cyclooctenylene (wherein the doublebond may be at an arbitrary site) groups.

The term a “divalent aromatic hydrocarbon group having 6 to 14 carbonatoms” as used in the formulae of Group C-1 and group C-2 herein meansan arylene or aralkylene group having 6 to 14 carbon atoms. Preferableexamples of the aromatic hydrocarbon group include phenylene, benzylene,phenethylene, naphthylene, anthrylene and phenanthrylene groups.

The term a “linear or branched aliphatic hydrocarbon group having 1 to 8carbon atoms and one or more hydroxyl groups” as used in the formulae ofGroup C-1 and Group C-2 herein means the linear or branched aliphatichydrocarbon group having 1 to 8 carbon atoms as described above whereinone or more carbon atoms at arbitrary sites carry hydroxyl groups. Whenit has two hydroxyl groups, these hydroxyl groups may be attached to asingle carbon atom.

The term a “linear or branched divalent aliphatic hydrocarbon grouphaving 1 to 8 carbon atoms and one or more hydroxyl groups” as used inthe formulae of Group C-1 and Group C-2 herein means the linear orbranched divalent aliphatic hydrocarbon group having 1 to 8 carbon atomsas described above wherein one or more carbon atoms at arbitrary sitescarry hydroxyl groups. When it has two hydroxyl groups, these hydroxylgroups may be attached to a single carbon atom.

The term a “linear or branched alicyclic hydrocarbon group having 1 to 8carbon atoms and one or more hydroxyl groups” as used in the formulae ofGroup C-1 and group C-2 herein means the linear or branched alicyclichydrocarbon group having 1 to 8 carbon atoms as described above whereinone or more carbon atoms at arbitrary sites carry hydroxyl groups. Whenit has two or more hydroxyl groups, these hydroxyl groups may beattached to a single carbon atom.

The term a “linear or branched aliphatic hydrocarbon group having 1 to 8carbon atoms and one or more ether groups” as used in the formulae ofGroup C-1 and Group C-2 herein means the linear or branched aliphatichydrocarbon group having 1 to 8 carbon atoms as described above whereinone or more ether groups are introduced into arbitrary sites.

The term a “linear or branched divalent aliphatic hydrocarbon grouphaving 1 to 8 carbon atoms and one or more ether groups” as used in theformulae of Group C-1 and Group C-2 herein means the linear or brancheddivalent aliphatic hydrocarbon group having 1 to 8 carbon atoms asdescribed above wherein one or more ether groups are introduced intoarbitrary sites.

The term a “linear or branched alicyclic hydrocarbon group having 1 to 8carbon atoms and one or more ether groups” as used in the formulae ofGroup C-1 and Group C-2 herein means the linear or branched alicyclichydrocarbon group having 1 to 8 carbon atoms as described above whereinone or more ether groups are introduced into arbitrary sites.

The term a “glycoside having 3 to 12 carbon atoms” as used in theformulae of Group C-1 and Group C-2 herein means an aldose or a ketoseformed by a glycoside bond and having 3 to 12 carbon atoms. Preferableexamples of the aldose or ketose include arabinose, lyxose, ribose,xylose, glucose, galactose, mannose and fructose.

The term a “linear or branched aliphatic hydrocarbon group having 1 to 8carbon atoms and a glycoside having 3 to 12 carbon atoms” as used in theformulae of Group C-1 and Group C-2 herein means the above-mentionedlinear or branched aliphatic hydrocarbon group having 1 to 8 carbonatoms wherein the above-mentioned glycoside having 3 to 12 carbon atomsis bonded via a glycoside bond to the arbitrary site.

The term a “linear or branched aliphatic hydrocarbon group having 1 to 8carbon atoms and one or more amide groups” as used in the formulae ofGroup C-1 and Group C-2 herein means the linear or branched aliphatichydrocarbon group having 1 to 8 carbon atoms as described above whereinone or more amide groups are introduced into arbitrary sites.

The term a “linear or branched aliphatic hydrocarbon group having 1 to 8carbon atoms and one or more carbonyl groups” as used in the formulae ofGroup C-1 and Group C-2 herein means the linear or branched aliphatichydrocarbon group having 1 to 8 carbon atoms as described above whereinone or more carbonyl groups are introduced into arbitrary sites.

The term a “linear or branched aliphatic hydrocarbon group having 1 to 8carbon atoms and one or more nitryl groups” as used in the formulae ofGroup C-1 and Group C-2 herein means the linear or branched aliphatichydrocarbon group having 1 to 8 carbon atoms as described above whereinone or more nitryl groups are introduced into arbitrary sites. When ithas two nitryl groups, these nitryl groups may be attached to a singlecarbon atom.

The polymer compound represented by the formula of Group C-1 and GroupC-2 according to the present invention can be produced in the followingmanner. First, one or more monomers serving as the starting material(s)of the polymer compound and a polymerization initiator are dissolved ina solvent and the polymerization reaction is initiated by, for example,heating. In this step, a bifunctional monomer (a crosslinking agent) maybe also dissolved in the solvent to give a crosslinked polymer. It isalso possible in this step to dissolve a chain transfer agent in thepolymerization solvent so as to control the molecular weight of thepolymer compound or to introduce a reactive functional group to theterminal of the polymer compound. After the completion of the polymerreaction, the polymer compound is not dissolved but re-precipitated fromthe solvent thereby giving the aimed temperature-responsive polymercompound.

The polymer compound represented by the formula of Group C-1 and GroupC-2 according to the present invention can be fixed onto the surface ofa support (silica gel, polymer gel, etc.) with the use of, for example,the reactive functional group having been introduced into the terminalthereof. Alternatively, a polymerization initiator, etc. is fixed ontothe surface of a solid (silica gel, polymer gel, etc.) and then one ormore monomers serving as the starting material(s) of the polymercompound are dissolved in a polymerization solvent. Next, thepolymerization reaction is started by, for example, heating in thepresence of the support (silica gel, polymer gel, etc.) carrying thepolymerization initiator thereon to thereby fix the polymer compoundonto the surface of the support (silica gel, polymer gel, etc.). In thiscase, a bifunctional monomer (a crosslinking agent) may be dissolved inthe solvent to give a crosslinked matter containing the polymercompound. It is also possible in this step to dissolve a chain transferagent in the polymerization solvent so as to control the molecularweight of the polymer compound or to introduce a reactive functionalgroup to the terminal of the polymer compound. Materials containing thethus obtained polymer compounds are applicable to adsorption andseparation materials such as various liquid chromatographic packings,drug carriers, dielectric and magnetic materials, piezoelectric andpyroelectric materials, degradable and reactive materials, biofunctionalmaterials, etc.

4. Compounds represented by the formula of Group D.

The polymer compound according to the present invention has thefollowing structure. Namely, a polymer material comprising a polymercompound consisting exclusively of a repeating unit represented by thefollowing formula (I) or a copolymer or a gel containing this unitstructure and showing temperature-responsiveness in a solution.

wherein Z represents a hydrogen atom or a methyl group; X represents ahydrogen atom or a linear or branched aliphatic hydrocarbon group having1 to 8 carbon atoms and carrying at least one hydroxyl group; Yrepresents a linear or branched aliphatic hydrocarbon group having 2 to8 carbon atoms and carrying at least one hydroxyl group, or X and Y mayform together a chemical bond; and n is an integer of 2 or more.

The term “linear or branched aliphatic hydrocarbon group having 1 to 8carbon atoms and carrying at least one hydroxyl group” as used in theformula of Group D in the present invention means a linear or branchedhydroxyl alkyl group having 1 to 8 carbon atoms, while the term “linearor branched aliphatic hydrocarbon group having 2 to 8 carbon atoms andcarrying at least one hydroxyl group” as used herein means a linear orbranched hydroxyalkyl group having 2 to 8 carbon atoms.

The term “X and Y form together a chemical bond” as used in the formulaof Group D in the present invention means that X and Y have each analiphatic hydrocarbon group and form together a chemical bond structurehaving 5 to 16 carbon atoms in total (i.e., X+Y) and at least onehydroxyl group. The term “X and Y form together a chemical bond” meansthat X and Y are covalently bonded to each other.

The hydroxyalkyl group has one or more hydroxyl groups. Preferableexamples of the hydroxyalkyl group include 1-hydroxypentyl,2-hydroxypentyl, 3-hydroxypentyl, trans-hydroxycyclohexyl,6-hydroxyhexyl, 2-hydroxy-3-methylpentyl, 5-hydroxy-3-ethylpentyl,3-hydroxyhexyl, 7-hydroxyheptyl, 6-hydroxyheptyl, 8,3-dihydroxyoctyl and8,5-dihydroxyoctyl groups.

Although n is not particularly restricted so long as it is an integer of2 or more, it preferably stands for a value giving a molecular weight ofnot more than 700,000, still preferably from 1,000 to 700,000,

The monomer represented by the formula of Group D is exemplified bycompounds which are synthesized by reacting acrylic acid chloride,methacrylic acid chloride, anhydrous acrylic acid or anhydrousmethacrylic acid with alkylamino alcohols. The “alkylamino alcohols”usable in the present invention are those having a linear or branchedaliphatic hydrocarbon group having 3 to 16 carbon atoms and at least onehydroxyl group or those having an alicyclic hydrocarbon group having acyclic structure with 3 to 16 carbon atoms and at least one hydroxylgroup. Preferable examples of the alkylamino alcohols include thosehaving 1 to 12 carbon atoms and at least one hydroxyl group such as4-aminopentanol, 5-aminopentanol, 3-aminopentanol, 2-aminopentanol,trans-aminopentanol, 6-aminohexanol andN-5-hydroxypentyl-N′-methyl-8-amino-3,5-dihydroxyoctyl.

The copolymer containing the above-mentioned repeating unit to be usedin the present invention means a random copolymer or a block copolymerof the monomer represented by the above chemical formula with othermonomers, for example, alkylacrylamides (t-butylacrylamide,n-butylacrylamide, i-butylacrylamide, acrylamide, hexylacrylamide,heptylacryl-amide, etc.), alkylmethacrylamides (t-butylmethacrylamide,n-butylmethacrylamide, butylmethacrylamide, hexylmethacryl-amide,heptylmethacrylamide, etc.), methacrylic acid, alkyl acrylates (n-butylacrylate, s-butyl acrylate, t-butyl acrylate, n-propyl acrylate,i-propyl acrylate, etc.), alkyl methacrylates (methyl methacrylate,n-butyl methacrylate, s-butyl methacrylate, t-butyl methacrylate,n-propyl methacrylate, i-propyl methacrylate, etc.), monomers havingfunctional groups such as hydroxyl, amino, sulfone and epoxy groups(hydroxyethyl methacrylate, hydroxyethylarcylamide,2-aminoethylmethacrylamide, aminostyrene, 2-(t-butylamino)-ethylmethacrylate, 2-sulfoethyl methacrylate, 3-sulfopropyl acrylate,glycidyl methacrylate, etc.) and styrene.

The term “gel structure” as used herein means one obtained by reactingthe polymer with a crosslinking agent such as methylene bisacrylamide.

The polymer compound represented by the formula of Group D according tothe present invention is produced in the following manner. One or moremonomers serving as the starting materials of the polymer compound and apolymerization initiator are dissolved in a polymerization solvent. Thenthe polymerization is initiated by, for example, heating. In this step,a bifunctional monomer may be dissolved to give a gel structurecontaining the desired polymer compound. It is also possible in thisstep to dissolve a chain transfer agent in the polymerization solventtoo so as to control the molecular weight of the polymer compound, or tointroduce a reactive functional group into the terminal of the polymercompound. After the completion of the polymerization reaction,re-precipitation is carried out in a solvent in which the desiredpolymer is insoluble. Thus, the stimulus-responsive polymer compound canbe obtained at the desired temperature.

The polymer compound represented by the formula of Group D according tothe present invention can be fixed onto the surface of a carrier (silicagel, polymer gel, etc.) with the use of, for example, a reactivefunctional group having been introduced into the terminal thereof.Alternatively, the polymer compound can be fixed on the surface of acarrier (silica gel, polymer gel, etc.) by fixing a polymerizationinitiator on the surface of a solid (silica gel, polymer, etc.),dissolving one or more monomers serving as the starting materials of thepolymer compound in a polymerization solvent, and then effecting apolymerization reaction, for example, under heating in the presence ofthe carrier (silica gel, polymer gel, etc.) having the polymerizationinitiator fixed thereon. Similar to the above-described case, abifunctional monomer may be dissolved in this step to give a gelstructure containing the desired polymer compound. It is also possiblein this step to dissolve a chain transfer agent in the polymerizationsolvent too so as to control the molecular weight of the polymercompound or to introduce a reactive functional group thereinto.Materials containing such a polymer compound are applicable to variousadsorption/separation carriers (liquid chromatographic packings,adsorbents, etc.), agents for releasing bioengineering products, etc.and biofunctional materials.

5. Compounds represented by the formulae of Group E-1, Group E-2, GroupE-3, Group E-4 and Group E-5.

wherein Z represents a methyl group or a hydrogen atom; n is an integerof 2 or more; X₁, X₂, X₃, X₄ and X₅ are the same or different and eachrepresents a hydrogen atom, a group R, or a group —CO—NH—R, providedthat at least one of X₁ to X₅ is a group —CO—NH—R (wherein R representsa linear or branched aliphatic hydrocarbon group having 1 to 6 carbonatoms, a linear or branched aliphatic hydrocarbon group having 1 to 10carbon atoms and containing at least one amide bond, a linear orbranched aliphatic hydrocarbon group having 1 to 10 carbon atoms andcontaining at least one hydroxyl group, an alicyclic hydrocarbon grouphaving 3 to 10 carbon atoms and containing at least one hydroxyl group,an alicyclic hydrocarbon group having 3 to 10 carbon atoms andcontaining at least one amide bond, or a hydrogen atom); and i is aninteger of from 0 to 6.

wherein n and m are each such an arbitrary value as to make n+m=1.0; Zrepresents a methyl group or a hydrogen atom; n is an integer of 2 ormore; X₁, X₂, X₃, X₄ and X₅ are the same or different and eachrepresents a hydrogen atom, a group R, or a group —CO—NH—R, providedthat at least one of X₁ to X₅ is a group —CO—NH—R (wherein R representsa linear or branched aliphatic hydrocarbon group having 1 to 6 carbonatoms, a linear or branched aliphatic hydrocarbon group having 1 to 10carbon atoms and containing at least one amide bond, a linear orbranched aliphatic hydrocarbon group having 1 to 10 carbon atoms andcontaining at least one hydroxyl group, an alicyclic hydrocarbon grouphaving 3 to 10 carbon atoms and containing at least one hydroxyl group,an alicyclic hydrocarbon group having 3 to 10 carbon atoms andcontaining at least one amide bond, or a hydrogen atom); i is an integerof from 0 to 6; Y represents an oxygen atom or a nitrogen atom; and R′represents a linear or branched aliphatic hydrocarbon group having 1 to6 carbon atoms, a linear or branched aliphatic hydrocarbon group having1 to 10 carbon atoms and containing at least one amide bond, a linear orbranched aliphatic hydrocarbon group having 1 to 10 carbon atoms andcontaining at least one hydroxyl group, an alicyclic hydrocarbon grouphaving 3 to 10 carbon atoms and containing at least one hydroxyl group,an alicyclic hydrocarbon group having 3 to 10 carbon atoms andcontaining at least one amide bond or a hydrogen atom.

wherein Z represents a methyl group or a hydrogen atom; n is an integerof 2 or more; X₁, X₂, X₃, X₄ and X₅ are the same or different and eachrepresents a hydrogen atom, a group R, or a group —CO—NH—R, providedthat at least one of X₁ to X₅ is a group —CO—NH—R (wherein R representsa linear or branched aliphatic hydrocarbon group having 1 to 6 carbonatoms, a linear or branched aliphatic hydrocarbon group having 1 to 10carbon atoms and containing at least one amide bond, a linear orbranched aliphatic hydrocarbon group having 1 to 10 carbon atoms andcontaining at least one hydroxyl group, an alicyclic hydrocarbon grouphaving 3 to 10 carbon atoms and containing at least one hydroxyl group,an alicyclic hydrocarbon group having 3 to 10 carbon atoms andcontaining at least one amide bond, or a hydrogen atom); A₁, A₂, A₃, A₄and A₅ are the same or different and each represents a carbon atom or anitrogen atom bonding to X_(n) (wherein n is an integer of 1 to 5)having a group —CO—NH—R or a group —CO—R, provided that at least one ofA₁ to A₅ is a nitrogen atom bonding to X_(n) (wherein n is an integer of1 to 5) having a group —CO—NH—R or a group —CO—R (wherein R is asdefined above); and i is an integer of from 0 to 6.

wherein n and m are each such an arbitrary value as to make n+m=1.0; Zrepresents a methyl group or a hydrogen atom; n is an integer of 2 ormore; X₁, X₂, X₃, X₄ and X₅ are the same or different and eachrepresents a hydrogen atom, a group R, or a group —CO—NH—R, providedthat at least one of X₁ to X₅ is a group —CO—NH—R (wherein R representsa linear or branched aliphatic hydrocarbon group having 1 to 6 carbonatoms, a linear or branched aliphatic hydrocarbon group having 1 to 10carbon atoms and containing at least one amide bond, a linear orbranched aliphatic hydrocarbon group having 1 to 10 carbon atoms andcontaining at least one hydroxyl group, an alicyclic hydrocarbon grouphaving 3 to 10 carbon atoms and containing at least one hydroxyl group,an alicyclic hydrocarbon group having 3 to 10 carbon atoms andcontaining at least one amide bond, or a hydrogen atom); A₁, A₂, A₃, A₄and A₅ are the same or different and each represents a carbon atom or anitrogen atom bonding to X_(n) (wherein n is an integer of 1 to 5)having a group —CO—NH—R or a group —CO—R, provided that at least one ofA₁ to A₅ is a nitrogen atom bonding to X_(n) (wherein n is an integer of1 to 5) having a group —CO—NH—R or a group —CO—R (wherein R is asdefined above); i is an integer of from 0 to 6; Y represents an oxygenatom or a nitrogen atom; and R′ represents a linear or branchedaliphatic hydrocarbon group having 1 to 6 carbon atoms, a linear orbranched aliphatic hydrocarbon group having 1 to 10 carbon atoms andcontaining at least one amide bond, a linear or branched aliphatichydrocarbon group having 1 to 10 carbon atoms and containing at leastone hydroxyl group, an alicyclic hydrocarbon group having 3 to 10 carbonatoms and containing at least one hydroxyl group, an alicyclichydrocarbon group having 3 to 10 carbon atoms and containing at leastone amide bond or a hydrogen atom.

wherein n is n is the number of the right kind of monomer unit comparedto the total number of the two kinds of monomer units shown and is anarbitrary value falling within the range 0.005≦n≦0.995; R¹ and R² arethe same or different and each represents a hydrogen atom or a methylgroup; X¹ and X² are the same or different and each represents an acidamide or ester group; Z¹, Z² and Z³ are the same or different and eachrepresents a hydrogen atom, a linear or branched aliphatic hydrocarbongroup having 1 to 8 carbon atoms, a linear or branched hydrocarbon grouphaving 1 to 8 carbon atoms and containing at least one hydroxyl group, alinear or branched hydrocarbon group having 1 to 8 carbon atoms andcontaining at least one ether group, a glycoside having 3 to 12 carbonatoms or a glycoside having 3 to 12 carbon atoms and containing a linearor branched hydrocarbon group having 1 to 8 carbon atoms, provided thatZ¹ or Z³ is a functional group carried by X¹ or X² which is a tertiaryamide; and Z⁴ represents a hydrogen atom, a hydroxyl group, an amidegroup, a linear or branched hydrocarbon group having 1 to 8 carbon atomsand containing at least one amide group, a linear or branchedhydrocarbon group having 1 to 8 carbon atoms and containing at least onecarbonyl group or a linear or branched hydrocarbon group having 1 to 8carbon atoms and containing at least one hydroxyl group which may beattached at an arbitrary position, i.e., o-, m- or p-position.

The term “linear or branched aliphatic hydrocarbon group having 1 to 6carbon atoms” as used in the formulae of Group E-1, Group E-2, GroupE-3, Group E-4 and Group E-5 in the present invention means a linear orbranched alkyl group having 1 to 6 carbon atoms, a linear or branchedalkenyl group having 1 to 6 carbon atoms or a linear or branched alkynylgroup having 1 to 6 carbon atoms. Among all, alkyl groups are preferabletherefor. Still preferable examples thereof include methyl, ethyl,n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl andn-hexyl groups.

The term “aliphatic hydrocarbon group having 1 to 10 carbon atoms andcontaining at least one amide bond” as used in the formulae of GroupE-1, Group E-2, Group E-3, Group E-4 and Group E-5 herein means a linearor branched aliphatic hydrocarbon group having 1 to 10 carbon atoms andcontaining one or more amide bonds at arbitrary positions.

The term “aliphatic hydrocarbon group having 1 to 6 carbon atoms andcontaining at least one hydroxyl group” as used in the formulae of GroupE-1, Group E-2, Group E-3, Group E-4 and Group E-5 herein means a linearor branched aliphatic hydrocarbon group having 1 to 10 carbon atoms andcontaining one or more hydroxyl groups at arbitrary positions.Preferable examples thereof include hydroxymethyl, 2-hydroxyethyl,3-hydroxypropyl, 5-hydroxyisopropyl, 4-hydroxybutyl, 5-hydroxypentyl,6-hydroxyhexyl, 5,7-dihydroxybutyl, 6,8-dihydroxyoctyl,5,9-dihydroxynonyl and 5,7,10-trihydroxydecyl groups.

The term “alicyclic hydrocarbon group having 3 to 10 carbon atoms andcontaining at least one hydroxyl group” as used in the formulae of GroupE-1, Group E-2, Group E-3, Group E-4 and Group E-5 herein means ahydroxycycloalkyl group having 3 to 10 carbon atoms. Preferable examplesthereof include 4-hydoxycyclohexyl, 1-methyl-4-hydroxycyclohexyl,2-hdyroxy-cyclopropyl and 3,5-dihdyroxycyclooctyl groups.

The term “alicyclic hydrocarbon group having 3 to 10 carbon atoms andcontaining at least one amide bond” as used in the formulae of GroupE-1, Group E-2, Group E-3, Group E-4 and Group E-5 herein means analicyclic group having 3 to 10 carbon atoms and containing one or moreamide groups at arbitrary positions.

The “cyclic secondary amine compound having an amide group” usable inthe formulae of Group E-1, Group E-2, Group E-3, Group E-4 and Group E-5herein for synthesizing the monomer represented by the formula of GroupE-1 means a cyclic secondary amine compound having 5 to 30 carbon atomsand containing one or more amide bonds or a cyclic secondary aminecompound having 5 to 30 carbon atoms and containing one or more hydroxylgroups and amide bonds. Preferable examples thereof include4-piperidinecarboxamide, N-methyl-4-piperidinecarboxamide,N-4-hydroxybutyl-4-piperidinecaroxamide,N-butyl-4-piperidine-carboxamide, 2-piperidinecarboxamide,N-methyl-2-piperidine-carboxamide andN,N′-dimethyl-2,4-piperidine-carboxamide.

The “cyclic secondary amine compound having at least one acyl bond”usable herein for synthesizing the monomer represented by the formula ofGroup E-3 means a cyclic secondary amine compound having 5 to 30 carbonatoms and containing one or more amide bonds or a cyclic secondary aminecompound having 5 to 30 carbon atoms and containing one or more acylbond. Preferable examples thereof include 1-acetylpiperazine,1-propionylpiperazine, 1-isobutyrylpiperazine, 1-hydroxybutylpiperazine,1-valerylpiperazine and 1-hydroxyvaleryl-piperazine.

The polymer compound represented by each of the formulae of Group E-1,Group E-2, Group E-3, Group E-4 and Group E-5 according to the presentinvention is produced in the following manner. One or more monomersserving as the starting materials of the polymer compound and apolymerization initiator are dissolved in a polymerization solvent. Thenthe polymerization is initiated by, for example, heating. In this step,a bifunctional monomer (i.e., a crosslinking agent) may be dissolved togive a crosslinked matter containing the desired polymer compound. It isalso possible to dissolve a chain transfer agent in the polymerizationsolvent too so as to control the molecular weight of the polymercompound or to introduce a reactive functional group into the terminalof the polymer compound. After the completion of the polymerizationreaction, re-precipitation is carried out in a solvent in which thedesired polymer is insoluble. Thus, the desired temperature-responsivepolymer compound can be obtained.

Functional groups (for example, carboxyl, hydroxyl, amino, nitryl,linear or branched alkyl having 1 to 20 carbon atoms and cyano groups)may be introduced into the polymer chain terminal of the polymercompound represented by the formula of Group E-1, Group E-2, Group E-3,Group E-4 or Group E-5 according to the present invention. Theintroduction can be carried out by conventionally known methods. Ifnecessary, it is possible in this step to use a chain transfer agent ora polymerization initiator. For example, use can be made therefor of achain transfer agent containing functional group(s) having 1 to 20carbon atoms (mercaptopropionic acid, aminoethanethiol, butanethiol,etc.) or a polymerization initiator containing amino and carboxylgroups.

The polymer compound represented by the formula of Group E-1, Group E-2,Group E-3, Group E-4 or Group E-5 according to the present invention canbe fixed onto the surface of a carrier (silica gel, polymer gel, etc.)with the use of, for example, a reactive functional group having beenintroduced into the terminal thereof. Alternatively, the polymercompound can be fixed on the surface of a carrier (silica gel, polymergel, etc.) by fixing a polymerization initiator on the surface of asolid (silica gel, polymer, etc.), dissolving one or more monomersserving as the starting materials of the polymer compound in apolymerization solvent, and then effecting a polymerization reaction,for example, under heating in the presence of the carrier (silica gel,polymer gel, etc.) having the polymerization initiator fixed thereon.Similar to the above-described case, a bifunctional monomer (i.e., acrosslinking agent) may be dissolved in this step to give a gelstructure containing the desired polymer compound. It is also possibleto dissolve a chain transfer agent in the polymerization solvent too soas to control the molecular weight of the polymer compound or tointroduce a reactive functional group thereinto. Materials containingsuch a polymer compound are applicable to various liquid chromatographicpackings, materials for the separation, adsorption or release ofbiological components (proteins, peptides, nucleic acids, etc.) andchemicals and biofunctional materials.

EXAMPLES

The present invention will be described in greater detail by referenceto the following Examples, but it should be understood that theinvention is not construed as being limited thereto.

Example A1 Synthesis of poly-acetamide-propyl methacrylate

0.9 g of 2-N-aminoethyl methacrylate hydrochloride was dissolved in amethanol solvent and 1.0 g of propionic anhydride and 0.9 g oftriethylamine (TEA) were added thereto. The resultant mixture wasstirred under ice-cooling for 4 hours. After the completion of thereaction, the solvent was distilled off with an evaporator and theprecipitate was filtered off. The filtrate was recovered and introducedinto a silica column. Thus, the eluate fraction containing the targetproduct was taken up and subjected to recrystallization to therebypurify a precursor of the target product acetamide-propyl methacrylate(CH₃CONH—(CH₂)₃—O—CO—C(CH₃)═CH₂) (yield: 75%). 0.3 g of this precursorwas dissolved in 5 ml of n-propanol and 6.2 mg of2,2-azobisisobutyronitrile (AIBN) was added thereto. Then, the mixturewas polymerized at 75° C. for 12 hours under a nitrogen atmosphere.After the completion of the polymerization, the reaction mixture wasice-cooled and a half of the solvent was removed with an evaporator. Theresidue was reprecipitated from an acetone solvent and dried in vacuo.Thus, the target product having a molecular weight (Mn) of 3,200,determined by gel permeation chromatography, was obtained. After drying,an aqueous solution of 0.1% by weight ofpoly-methacryloyl-acetylaminoethyl-ester was prepared and the change inthe permeability of the solution depending on temperature was measured(FIG. 1).

Example A2 Synthesis of poly-propionamide-propyl acrylate

4 g of 3-aminopropyl alcohol was dissolved in 100 ml of adichloromethane solvent and 10 g of propionic anhydride and 0.9 g oftriethylamine (TEA) were added thereto. The resultant mixture wasstirred at 50° C. for 4 hours. After the completion of the reaction, thesolvent was distilled off with an evaporator and the precipitate wasfiltered off. The filtrate was recovered and introduced into a silicacolumn. Thus, the eluate fraction containing the target product wastaken up and subjected to recrystallization to thereby purify aprecursor of the target product propionamide-propyl acrylate(CH₃CH₂CONH—(CH₂)₃—O—CO—CH═CH₂). 1.0 g of this precursor and 8 mg of2,2-azobis(2-amidinopropane)dihydrochloride were dissolved in 10 ml ofethanol and polymerized at 70° C. for 3 hours. After the completion ofthe polymerization, an adequate amount of the solvent was removed withan evaporator. The residue was reprecipitated from an alcohol/ethylacetate/acetone solvent to give poly-propionamide-acrylate. An aqueoussolution of 1% by weight of this polymer was prepared and introducedinto a thermostat at 90° C. Then, the solution became cloudy. Next, thecloudy solution was ice-cooled. As a result, the solution becametransparent. Since these phenomena occurred reversibly, it was confirmedthat this polymer showed temperature-responsiveness in the aqueoussolution.

Example B1 Synthesis of propionamide-propyl methacrylamide(CH₃CH₂CONH—(CH₂)₃—NH—CO—C(CH₃)═CH₂)

0.8 g of 3-aminopropyl methacrylamide hydrochloride was dissolved in amethanol solvent and 1.0 g of propionic anhydride and 0.9 g oftriethylamine (TEA) were added thereto. The resultant mixture wasstirred under ice-cooling for 4 hours. After the completion of thereaction, the solvent was distilled off with an evaporator and theprecipitate was filtered off. The filtrate was recovered and introducedinto a silica column. Thus, the eluate fraction containing the targetproduct was taken up and subjected to recrystallization to therebypurify the target propionamide-propyl methacrylamide (yield: 95%). FIG.2 shows the result of the mass spectrometry of the purified productwhile FIG. 3 shows the result of ¹H-NMR thereof.

Example B2 Synthesis of acetylamide-propyl methacrylamide(CH₃CONH—(CH₂)₃—NH—CO—C(CH₃)═CH₂)

0.8 g of 3-aminopropyl methacrylamide hydrochloride was dissolved in amethanol solvent and 0.9 g of acetic anhydride and 0.9 g oftriethylamine (TEA) were added thereto. The resultant mixture wasstirred under ice-cooling for 4 hours. After the completion of thereaction, the solvent was distilled off with an evaporator and theprecipitate was filtered off. The filtrate was recovered and introducedinto a silica column. Thus, the eluate fraction containing the targetproduct was taken up and subjected to recrystallization to therebypurify the target acetylamide, N-3-propyl methacrylamide (yield: 92%).

Example B3 Synthesis of butyrylamide-propyl methacrylamide(CH₃(CH₂)₂CONH—(CH₂)₃—NH—CO—C(CH₃)═CH₂)

0.8 g of 3-aminopropyl methacrylamide hydrochloride was dissolved in amethanol solvent and 1.5 g of butyric anhydride and 0.9 g oftriethylamine (TEA) were added thereto. The resultant mixture wasstirred under ice-cooling for 4 hours. After the completion of thereaction, the solvent was distilled off with an evaporator and theprecipitate was filtered off. The filtrate was recovered and introducedinto a silica column. Thus, the eluate fraction containing the targetproduct was taken up and subjected to recrystallization to therebypurify the target butyrylamide, N-3-propyl methacrylamide (yield: 92%).

Example B4 Synthesis of propionamide-propyl methacrylamide(CH₃CH₂CONH—(CH₂)₃—NH—CO—CH═CH₂)

5.0 g of 1,3-aminopropyldiamine was mixed with 150 ml of an acetonitrilesolvent under ice-cooling. Then, a solution of 6.0 g of acrylic acidchloride dissolved in 60 ml of acetonitrile was slowly dropped thereintounder-ice cooling with stirring for hours. After the completion of thestirring, the precipitate was recovered and dissolved in a TEA/methanolsolvent. Then 3-aminorpopyl acrylamide was purified with a silicacolumn. To the eluate containing this product, 10 g of propionicanhydride was added and the resultant mixture was stirred under-icecooling. After the completion of the reaction, the solvent was distilledoff with an evaporator and the precipitate was filtered off. Thefiltrate was recovered and introduced into a silica column. Thus, theeluate fraction containing the target product was taken up and subjectedto recrystallization to thereby purify the target propionamide-propylmethacrylamide (yield: 75%).

Example B5 Polymerization of butyrylamide-propylmethacryl-amide

0.3 g of the monomer butylamide, N-3-propyl methacrylamide was dissolvedin 5 ml of n-propanol. After adding 6.2 mg of 2,2-azobisisobutyronitrile(AIBN), the mixture was polymerized at 75° C. for 12 hours under anitrogen atmosphere. After the completion of the polymerization, a halfof the solvent was removed with an evaporator and the residue wasreprecipitated from an acetone solvent and dried in vacuo. Next, anaqueous solution of 1% by weight of polybutylamide andN-3-propylmethacrylamide was prepared and the permeability of thesolution was measured at various temperatures. Based on these results,it was confirmed that the polymer showed a cloud point in the aqueoussolution. FIG. 4 shows the results.

Example B6 Polymerization of acetylamide-propylmethacryl-amide

0.4 g of the monomer acetylamide, N-3-propyl methacrylamide wasdissolved in 5 ml of methanol. After adding 6.0 mg of2,2-azobis(2-amidinopropane)dihydrochloride, the mixture was polymerizedat 65° C. for 4 hours under a nitrogen atmosphere. After the completionof the polymerization, the reaction mixture was ice-cooled and a half ofthe solvent was removed with an evaporator. The residue wasreprecipitated from an acetone solvent and dried in vacuo. Next, anaqueous solution of 1% by weight of polyacetylamide and N-3-propylmethacrylamide and NaCl solutions (1.0, 0.5 and 0.25 M) thereof wereprepared and the permeabilities of these solutions were measured atvarious temperatures. Based on these results, it was confirmed that thepolymer showed cloud points in the NaCl solutions and the cloud pointwas lowered with an increase in the salt concentration. FIG. 5 shows theresults.

Example B7 Polymerization of propionamide-propylmethacrylamide

0.7 g of propionamide-propyl methacrylamide was dissolved in 8 ml ofmethanol. After adding 6.2 mg of2,2-azobis(2-amidinopropane)dihydrochloride, the mixture was polymerizedat 65° C. for 4 hours under a nitrogen atmosphere. After the completionof the polymerization, the reaction mixture was ice-cooled and a half ofthe solvent was removed with an evaporator. The residue wasreprecipitated from an acetone solvent and dried in vacuo. Next, asolution of 1% by weight of polyacetylamide-propyl methacrylamide in a500 mM aqueous solution of NaCl was prepared and introduced into athermostat at 70° C. Thus, the solution became cloudy. Next, the cloudysolution was ice-cooled. As a result, the polymer was dissolved. Sincethese phenomena occurred reversibly, it was confirmed that this polymerhad a cloud point. Further, an aqueous solution of 1% by weight ofpolyacetylamide-propyl methacrylamide and NaCl solutions (0.5 and 0.25M) thereof were prepared and the permeabilities of these solutions weremeasured at various temperatures. Based on these results, it wasconfirmed that the polymer showed cloud points in the NaCl solutions andthe cloud point was lowered with an increase in the salt concentration.FIG. 6 shows the results.

Example C1

3-Aminoacetanilide (50 mmol) and triethylamine (56 mmol) were dissolvedin dimethylformamide (100 mL) and acryloyl chloride (55 mmol) wasdropped thereinto under ice-cooling. After the completion of theaddition, the resultant mixture was stirred at room temperature for 2hours. After filtering off the precipitate, the solvent was distilledoff. Then the solid matter thus obtained was recrystallized from asolvent mixture of hexane, ethyl acetate and acetone to give3-acrylamido-acetanilide at a yield of 61%. 4-Aminoacetanilide (50 mmol)and triethylamine (56 mmol) were dissolved in dimethylformamide (100 mL)and acryloyl chloride (55 mmol) was dropped thereinto under ice-cooling.After the completion of the addition, the resultant mixture was stirredat room temperature for 2 hours. After filtering off the precipitate,the solvent was distilled off. Then the solid matter thus obtained wasrecrystallized from a solvent mixture of water with methanol to give4-acrylamidoacetanilide at a yield of 77%.

4-Aminobenzamide (29 mmol) and triethylamine (33 mmol) were dissolved indimethylformamide (100 mL) and acryloyl chloride (33 mmol) was droppedthereinto under ice-cooling. After the completion of the addition, theresultant mixture was stirred at room temperature for 2 hours. Afterfiltering off the precipitate, the solvent was distilled off. Then thesolid matter thus obtained was recrystallized from a solvent mixture ofwater with methanol to give 4-acrylamidobenzamide at a yield of 60%.

Acrylamide was dissolved in water and subjected to radicalpolymerization at 70° C. with the use of2,2′-azobis(2-amidinopropane)dihydrochloride as an initiator to therebygive polyacrylamide as a homopolymer. Hydroxymethyl acrylamide wasdissolved in water and subjected to radical polymerization at 70° C.with the use of 2,2′-azobis(2-amidinopropane)dihydrochloride as aninitiator to thereby give polyhydroxymethyl acrylamide as a homopolymer.N,N-Dimethyl acrylamide was dissolved in water and subjected to radicalpolymerization at 70° C. with the use of2,2′-azobis(2-amidinopropane)dihydrochloride as an initiator to therebygive poly(N,N-dimethyl acrylamide) as a homopolymer. Glycerolmonomethacrylate was dissolved in water and subjected to radicalpolymerization at 70° C. with the use of2,2′-azobis(2-amidinopropane)dihydrochloride as an initiator to therebygive polyglycerol monomethacrylate as a homopolymer. Glycosyloxyethylmethacrylate was dissolved in water and subjected to radicalpolymerization at 70° C. with the use of2,2′-azobis(2-amidinopropane)dihydrochloride as an initiator to therebygive polyglycosyloxyethyl methacrylate as a homopolymer.3-Acrylamidoacetanilide was dissolved in dimethylformamide and subjectedto radical polymerization at 70° C. with the use ofazobisisobutyronitrile as an initiator to thereby givepoly(3-acrylamidoacetanilide) as a homopolymer. 4-Acrylamidoacetanilidewas dissolved in dimethylformamide and subjected to radicalpolymerization at 70° C. with the use of azobisisobutyronitrile as aninitiator to thereby give poly(4-acrylamidoacetanilide) as ahomopolymer. 4-Acrylamido-benzamide was dissolved in dimethylformamideand subjected to radical polymerization at 70° C. with the use ofazobisisobutyronitrile as an initiator to thereby givepoly(4-acrylamidobenzamide) as a homopolymer.

The solubility in water of each of these 8 homopolymers was examined.Although polyacrylamide, polyhydroxymethyl acrylamide,polyglycosyloxyethyl methacrylate, polyglycerol monomethacrylate andpoly(N,N-dimethyl acrylamide) were soluble in water, they showed nochange in turbidity, etc. with a temperature change, thereby expressingno temperature-responsiveness. On the other hand,poly(3-acrylamido-acetanilide), poly(4-acrylamidoacetanilide) andpoly(4-acrylamidobenzamide) were hardly soluble in water and showed noremarkable change in solubility with a temperature change, therebyexpressing no temperature-responsiveness.

Example C2

Acrylamide (0.90 or 0.85 mmol), 3-acrylamidoacetanilide (0.1 or 0.15mmol) and azobisisobutyronitrile (0.05 mmol) were dissolved in a solventmixture of dimethylformamide (2.5 mL) with dimethyl sulfoxide (2.5 mL)and subjected to radical polymerization at 70° C. After reprecipitatingfrom ether, poly(acrylamide-co-3-acrylamidoacetanilide) was obtained.

Each copolymer thus obtained was dissolved in water to give aconcentration of 1% by weight and a change in the turbidity depending ontemperature was observed at 500 nm by using a spectrophotometer. FIG. 7shows the results. Thus, it was confirmed that the copolymer obtained byfeeding acrylamide and 3-acrylamidoacetanilide at a ratio of 90:10(mol/mol) was a temperature-responsive polymer compound having an UCSTof 9° C. while the one obtained by feeding acrylamide and3-acrylamidoacetanilide at a ratio of 95:15 (mol/mol) was atemperature-responsive polymer compound having an UCST of 69° C. it wasalso confirmed that the temperature temperature-responsiveness could bechanged by altering the feeding ratio.

Further aqueous solutions containing 0.1, 0.5, 1.0 and 3.0% by weight ofthe copolymer obtained by feeding acrylamide and 3-acrylamidoacetanilideat a ratio of 85:15 were prepared and the UCSTs thereof were measured.As a result, these aqueous solutions respectively showed UCSTs of 32°C., 66° C., 69° C. and 72° C., thus showing that UCST would varydepending on the concentration of the polymer compound too.

Example C3

Acrylamide (0.85 mmol), 3-acrylamidoacetanilide (0.15 mmol),3-mercaptopropionic acid (10 μmol, 5 μmol or 0 μmol) and2,2′-azobis(4-cyanovaleric acid) (0.01 mmol) were dissolved in a solventmixture of dimethylformamide (2.5 mL) with dimethyl sulfoxide (2.5 mL)and subjected to radical polymerization. After reprecipitating fromether, poly(acrylamide-co-3-acrylamidoacetanilide) copolymers withdifferent molecular weights were obtained.

The number-average molecular weights of these copolymers measured by theterminal analysis method were 6500, 9300 and 14200 respectively. Eachcopolymer was dissolved in water to give a concentration of 1.0% byweight and the temperature-responsiveness was determined. As a result,these copolymers had UCSTs of 26° C., 38° C. and 72° C. respectively.Thus, it was also confirmed that the UCST of a polymer compound could becontrolled depending on the molecular weight.

Example C4

Acrylamide (0.85 mmol), 3-acrylamidoacetanilide (0.15 mmol),N,N′-methylenebisacrylamide (0.10 mmol) and2,2′-azobis(2-amidinopropane)dihydrochloride (0.05 mmol) were dissolvedin water (10 mL) and subjected to radical polymerization at 70° C. togive a crosslinked product containingpoly(acrylamide-co-3-acrylamidoacetanilide).

Then it was observed whether an aqueous solution of this crosslinkedproduct would undergo a change in turbidity depending on temperature ornot. Thus, it was confirmed that the crosslinked product was atemperature-responsive one having an UCST which was cloudy underice-cooling and soluble at 90° C.

Example C5

Acrylamide (0.8 mmol), 4-acrylamidoacetanilide (0.2 mmol) and2,2′-azobis(2-amidinopropane)dihydrochloride (0.05 mmol) were dissolvedin water (5 mL) and subjected to radical polymerization at 70° C. togive poly(acrylamide-co-4-acrylamidoacetanilide).

Then the polymer compound was dissolved in water and a change in theturbidity thereof depending on temperature was observed. Thus, it wasconfirmed that this polymer compound was a temperature-responsive onehaving an UCST which was cloudy under ice-cooling and soluble at 90° C.

Example C6

N,N-Dimethyl acrylamide (0.9 mmol), 3-acrylamido-acetanilide (0.1 mmol)and 2,2′-azobis(2-amidinopropane)dihydrochloride (0.05 mmol) weredissolved in water (5 mL) and subjected to radical polymerization at 70°C. to give poly(N,N-dimethylacrylamide-co-4-acrylamido-acetanilide).

Then the polymer compound was dissolved in water and a change in theturbidity thereof depending on temperature was observed. Thus, it wasconfirmed that this polymer compound was a temperature-responsive onehaving an UCST which was soluble under ice-cooling and cloudy at 90° C.

Example C7

Hydroxymethyl acrylamide (0.87 mmol), 3-acrylamido-acetanilide (0.13mmol) and 2,2′-azobis(2-amidinopropane)dihydrochloride (0.05 mmol) weredissolved in water (5 mL) and subjected to radical polymerization at 70°C. to give poly(hydroxymethylacrylamide-co-3-acrylamidoacetanilide).

Then the polymer compound was dissolved in water and a change in theturbidity thereof depending on temperature was observed. Thus, it wasconfirmed that this polymer compound was a temperature-responsive onehaving an UCST which was cloudy under ice-cooling and soluble at 90° C.

Example C8

Acrylamide (0.85 mmol), 4-acrylamidobenzamide (0.15 mmol) and2,2′-azobis(2-amidinopropane)dihydrochloride (0.05 mmol) were dissolvedin a solvent mixture of dimethylformamide (2.5 mL) with dimethylsulfoxide (2.5 mL) and subjected to radical polymerization at 70° C. togive poly(acrylamide-co-4-acrylamidobenzamide).

Then the polymer compound was dissolved in water to give a concentrationof 3.0% by weight and temperature was changed. As a result, it was foundout that this compound was a temperature-responsive polymer compoundhaving an UCST of 46° C. Since an aqueous solution (3.0% by weight) ofthe copolymer obtained by feeding acrylamide and 3-acrylamidoacetanilideat a ratio of 85:15 showed an UCST of 72° C., it was found out that thetemperature-responsiveness was affected by functional groups.

Example C9

Glycerol monomethacrylate (0.7 mmol), 4-acrylamido-benzamide (0.3 mmol)and 2,2′-azobis(2-amidinopropane)dihydrochloride (0.05 mmol) weredissolved in water (5 mL) and subjected to radical polymerization at 70°C. to give poly(glycerol monomethacrylate-co-4-acrylamido-benzamide).

Then the polymer compound was dissolved in water and a change in theturbidity thereof depending on temperature was observed. Thus, it wasconfirmed that this polymer compound was a temperature-responsive onehaving an UCST which was cloudy under ice-cooling and soluble at 90° C.

Example C10

Glycosyloxyethyl methacrylate (7.7 mmol), 4-acrylamido-benzamide (5.1mmol), 3-mercaptopropionic acid (0.4 mmol) and2,2′-azobis(4-cyanovaleric acid) (0.3 mmol) were dissolved indimethylformamide (30 mL). After subjected to radical polymerization at70° C. and reprecipitating from dioxane, poly(glycosyloxyethylmethacrylate-co-4-acrylamidebenzamide) was obtained. It was confirmedthat the copolymer had a number-average molecular weight of 8000 bymeasuring by GPC (gel permeation chromatograph) with the use as a mobilephase of dimethylformamide containing 10 mM of lithium bromide. Also, itwas confirmed that the copolymer had a number-average molecular weightof 7000 by the terminal analysis method by using 10 mM sodium hydroxide.Moreover, it was confirmed that this polymer compound carried a terminalcarboxyl group which was a reactive functional group. It was alsoconfirmed by ¹H-NMR that the copolymer compound obtained above containedrepeating units of glycosyloxyethyl methacrylate and4-acrylamido-benzamide at a composition ratio of 62:38 (mol/mol).

The copolymer thus synthesized was dissolved in water and a change inthe turbidity thereof depending on temperature was observed at 500 nm byusing a spectrophotometer. FIG. 8 shows the results. Thus it wasconfirmed that this copolymer was also a temperature-responsive polymercompound having an UCST of 35° C.

The polymer thus synthesized (0.8 g) and a condensing agent EEDQ (30 mg)were dissolved in dimethylformamide (15 mL). To the obtained solution,aminopropylsilyl silica gel (0.8 g) was added and the resultant mixturewas stirred for 24 hours to thereby fix the copolymer on the silica gel.This silica gel was packed into a stainless steel column having an innerdiameter of 4.6 mm and a height of 30 mm.

By using water as a mobile phase, the relative retentions of cortisoneacetate in this column were determined at various temperatures. FIG. 10shows the results. The slope of the graph shows a large change at around35° C., which indicates that the copolymer underwent a structural changeat around this temperature and thus the retention of the solute (i.e.,cortisone acetate) was affected thereby. Thus, it has been found outthat the elution behaviors of the solute can be controlled by using theabove-described copolymer as a separation material such as achromatographic support.

Example C11

Acrylic acid (20 g) and 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide(30 g) were dissolved in water (300 mL). Then aminohydroxybutyric acid(2.0 g) was added to the obtained solution and reacted at roomtemperature for 10 hours. After the completion of the reaction, waterwas distilled off and the residue was sufficiently washed with hexane(200 mL) in 3 portions. Next, this residue was dissolved indimethyl-formamide (350 mL), in which N-hydroxysuccinimide (3 g) andN,N′-dicyclohexylcarbodiimide (3 g) had been dissolved, and reactedovernight. After the completion of the reaction, the solvent wasdistilled off. Ethyl acetate was added to the residue thus obtained andthe precipitate thus formed was filtered off. The filtrate was recoveredand a fraction containing the target product was taken up by silica gelcolumn chromatography with the use of ethyl acetate as a mobile phaseand alumina column chromatography. Next, a half of the solvent wasdistilled off with an evaporator and recrystallization was carried outby adding a hexane solvent at −20° C. to thereby give the targetmonomers (N-propylacetamide, N-propyl acrylamide) (yield: 68%).

N-Propylacetamide and N-propyl acrylamide (0.8 g) and2,2′-azobis(4-cyanovaleric acid) (3 mg) were dissolved indimethylformamide (8 mL). After purging with nitrogen, the mixture waspolymerized in a sealed container at 65° C. for 4 hours. After thecompletion of the reaction, the solvent was distilled off and theresidue was reprecipitated from acetone to give poly(N-propylacetamide,N-propyl acrylamide) (yield: 83%).

Then this polymer compound was dissolved in water and a change in theturbidity thereof depending on temperature was observed. Thus, it wasconfirmed that this polymer compound was a temperature-responsive onehaving an LCST which was soluble under ice-cooling and cloudy at 90° C.

Example D1

5-Aminopentanol (5.4 g) and triethylamine (5.6 ml) were added todimethylformamide (140 ml). Into the resultant solution, a solution ofacrylic acid chloride (4.2 ml) in dimethylformamide (30 ml) was droppedat −40° C. under stirring. After 2 hours, the solution was filtered andthe precipitate was eliminated. Next, the solvent was eliminated withthe use of a rotary evaporator. After the elimination of the solvent,the residue was dissolved in acetone and an eluate containing the targetproduct was taken up with the use of a silica gel column. Then thesolvent was eliminated with a vacuum pump to purify5-hydroxypentylacrylamide (2.1 g).

The obtained 5-hydroxypentylacrylamide (1.0 g) and4,4′-azobis(4-cyanovaleric acid) (20 mg) employed as a polymerizationinitiator were added to dimethylformamide (5 ml). After degassing by thefreezing-thawing method, radical polymerization was carried out at 65°C. for 3 hours. After the completion of the polymerization reaction,dimethyl sulfoxide (7 ml) was added to the liquid reaction mixture. Theobtained mixture was subjected to re-precipitation in a mixture ofacetone/ether (volume ratio: 1:3) to thereby give the target homopolymerof poly(5-hydroxypentylacrylamide) (0.51 g). The number-averagemolecular weight (Mn) of the obtained polymer was 20,000 and themolecular weight distribution (Mw/Mn) thereof was 2.77.

This homopolymer was dissolved in an aqueous solution to give aconcentration of 0.1% by weight. Then the change in transmittance with achange in temperature was monitored (FIG. 10). Thus, it was indicatedthat the cloud point of this polymer was about 40.8° C.

To fix this polymer on a chromatographic packing, this polymer (0.3 g),hydroxysuccinimide (0.8 g) and dicyclo-hexylcarbodiimide (0.8 g) weredissolved in dimethylformamide (30 ml) and stirred at room temperatureovernight. After the completion of stirring, the precipitate thus formedwas sufficiently removed and re-precipitation was effected in a solventmixture of acetone/diethyl ether (volume ratio: 1:3). Aftervacuum-drying, the desired polymer carrying a hydroxysuccinimide groupat the polymer terminal was obtained. Next, this activated polymer (0.15g) and aminopropyl silica (0.75 g) were added to dimethylformamide (30ml) and thus the polymer was fixed on the silica. After the fixation,the above procedure was repeated under the same conditions to therebyfix the polymer. The elemental analysis data indicated that the organicmatter content was increased by 10% by weight, which proved that thepolymer had been fixed.

This silica was packed in a stainless column (4.6×30 mm) and peptideswere separated by using the same. As a result, it was proved that theretention times of peptides (β-endorphin, angioten, etc.) in an aqueousmobile phase varied depending on temperature.

Example D2

Trans-aminocyclohexanol (5.0 g) and triethylamine (6.1 ml) were added tochloroform (100 ml). Into the obtained mixture, a solution of acrylicacid chloride (3.4 ml) in chloroform (30 ml) was dropped underice-cooling over 2 hours, thus effecting a reaction. After thecompletion of the dropping/reaction, the resultant mixture was stirredat room temperature for 1 hour. Subsequently, the solvent was eliminatedby using a rotary evaporator. After the elimination of the solvent,ethyl acetate (250 ml) was added to the residue and the precipitate thusformed was filtered off. Next, the filtrate was treated with a silicacolumn and an eluate containing the target product was taken up. Afterconcentrating, n-hexane (250 ml) was added thereto followed byrecrystallization, filtration and drying, thus giving the target4-hydroxycyclohexylacrylamide (2.4 g).

This monomer (0.8 g) and 4,4′-azobis(4-cyanovaleric acid) (5 mg)employed as a polymerization initiator were added to dimethylformamide(5 ml). After degassing by the freezing-thawing method, polymerizationwas carried out at 65° C. for 3 hours. After the completion of thepolymerization, dimethyl sulfoxide (7 ml) was added to the liquidreaction mixture. The obtained mixture was subjected to re-precipitationin a mixture of acetone/diethyl ether (volume ratio: 1:3) followed byvacuum-drying to thereby give the target homopolymer ofpoly(trans-hydroxycyclohexylacrylamide) (0.90 g). Based on the GPC data,the number-average molecular weight (Mn) of the obtained polymer wasestimated as 28,000 and the molecular weight distribution (Mw/Mn)thereof was 2.68. By monitoring the transmittance with a change intemperature (FIG. 11), it was clarified that the cloud point of thispolymer was about 41.2° C. The number average molecular weightdetermined by the terminal titration method was also the same as thevalue described above.

Example D3

5-Hydroxypentylacrylamide (1.0 g), t-butylacrylamide (0.12 g) and2,2′-azobisisobutyronitrile (12 mg) were dissolved in dimethyl sulfoxide(6 ml) and polymerization was carried out at 70° C. for 3 hours. Afterthe completion of the polymerization reaction, the reaction mixture wasadded to a mixture of acetone/ether (volume ratio: 1:3) to give thetarget copolymer. This copolymer was dissolved in an aqueous solution togive a concentration of 1% by weight and then observation was made whilechanging temperature to examine whether it showed a cloud point or not.As a result, this polymer was dissolved under ice-cooling but becamecloudy at 90° C., thus showing a cloud point.

Example D4

6-Aminohexanol (2.0 g) and triethylamine (2.1 mg) were dissolved in achloroform solvent (80 ml). Into the obtained solution, a solution ofacrylic acid chloride (1.4 ml) in chloroform (20 ml) was dropped underice-cooling over 3 hours. After the completion of the addition, themixture was stirred at room temperature for 3 hours. After eliminatingthe solvent with the use of an evaporator, a precipitate was obtained.Then ethyl acetate (120 ml) was added thereto and the precipitate wasfiltered off. The filtrate was concentrated by using an evaporator andthen treated with a silica column to take up an eluate containing thetarget product followed by concentration and recrystallization to givethe desired 6-hydroxy-hexyl-acrylamide (1.4 g). This6-hydroxyhexyl-acrylamide (1.0 g) and 2,2′-azobisisobutylnitrile (10 mg)employed as a polymerization initiator were dissolved indimethylformamide (5 ml). After degassing by the freezing-thawingmethod, polymerization was carried out at 70° C. for 2 hours. After thecompletion of the polymerization, the polymer was re-precipitated byusing a solvent mixture tetrahydro-furan/ether (1:1) followed byvacuum-drying to give the target poly(6-hydroxyhexyl-acrylamide) (0.84g). Based on the GPC data, the number-average molecular weight (Mn) ofthe obtained polymer was estimated as 9,600 and the molecular weightdistribution (Mw/Mn) thereof was 4.05.

This polymer was dissolved in an aqueous solution to give aconcentration of 1% by weight and the change in transmittance wasmonitored while changing temperature (FIG. 12). To examine thetemperature-responsiveness in an aqueous solution of guanidine, thepolymer was dissolved in a 1.0 M aqueous solution of guanidinehydrochloride to give the same concentration and the change intransmittance was monitored while changing temperature. As a result, itwas clarified that this polymer showed a cloud point at 2.1° C. in theaqueous solution and at 5.2° C. in the 1.0 M aqueous solution ofguanidine hydrochloride.

Example D5

5-Hydroxypentylacrylamide (0.8 g), acrylamide (0.2 g) and2,2′-azobizisobutylnitrile (10 mg) were dissolved in dimethyl sulfoxide(7 ml) and polymerization was performed at 70° C. for 2 hours. After thecompletion of the polymerization reaction, the mixture was added to asolvent mixture of acetone/ether (volume ratio: 1:3) to give the targetcopolymer. This copolymer was dissolved in an aqueous solution to give apolymer concentration of 1% by weight. Then, observation was made whilechanging temperature to examine whether it showed a cloud point or not.As a result, this polymer was dissolved under ice-cooling but becamecloudy at 90° C., thus showing a cloud point.

Example E1

N-Methacryloyl-N′-benzoyl-1,3-diaminopropane (2.8 mg),N-acryloyl-N′-4-piperidinecarboxamide (6.1 mg) and2,2′-azobis(1-amidinopropane)dihydrochloride (7 mg) were dissolved inwater (1 ml) and subjected to radical polymerization at 80° C. to give acopolymer. Then, this polymer was dissolved in water and the change inturbidity was monitored while changing temperature. As a result, it wascloudy under ice-cooling but dissolved at 90° C., which indicated thatit was a temperature-responsive polymer having the UCST. When guanidinehydrochloride was added to this aqueous solution, the phenomenon of theUCST disappeared.

Example E2

N-Methacryloyl-N′-hexanoyl-1,3-diaminopropane (4.7 mg),N-acryloyl-N′-4-piperidinecarboxamide (20.1 mg) and2,2′-azobis(1-amidinopropane)dihydrochloride (4.8 mg) were dissolved inwater (2.5 ml) and subjected to radical polymerization at 80° C. to givea copolymer. Then, this polymer was dissolved in water and the change inturbidity was monitored while changing temperature. As a result, it wasdissolved under ice-cooling but cloudy at 90° C., which indicated thatit was a temperature-responsive polymer having the UCST.

Example E3

Into a liquid reaction mixture of 4-piperidine-carboxamide (5.0 mg) andtriethylamine (5.3 ml) in dimethylformamide (70 ml), a solution ofacrylic acid (3.2 ml) dissolved in dimethylformamide (20 ml) was droppedunder ice-cooling over 3 hours. After the completion of the addition,the resultant mixture was stirred at room temperature for 1 hour andthen filtered. From the filtrate thus obtained, the solvent waseliminated by using a rotary evaporator. After adding an acetonesolution to the residue, the mixture was treated with a silica gelcolumn to give N-acryloyl-4-piperidinecarboxamide (1.4 g).

N-Acryloyl-4-piperidinecarboxamide (0.94 g) and2,2′-azobis(4-cyanovaleric acid) (50 mg) were dissolved indimethylformamide (6 ml) and polymerized at 70° C. Subsequently, thereaction mixture was re-precipitated in a solvent mixture ofethanol/diethyl ether (1:4) and the precipitate was taken up byfiltration and vacuum-dried. Thus, the targetpoly(4-piperidinecarboxamide) (0.92 g) was obtained.

This polymer was dissolved in a 200 mM aqueous solution of ammoniumsulfate, a 300 mM aqueous solution of ammonium sulfate and a 500 mMaqueous solution of ammonium sulfate each to give a polymerconcentration of 1% by weight and changes in turbidity with temperaturechange (FIG. 13) and changes in turbidity under elevating and loweringtemperature (FIG. 14) were compared. Thus, it was confirmed that thispolymer showed the UCST in the aqueous ammonium sulfate solutions. Itwas also found that the UCST shifted toward the high temperature sidewith an increase in the salt concentration. Next, this polymer (0.9 g)was treated with hydroxysuccinimide (0.6 g) and dicyclohexylcarbodiimide(0.5 g) in a solvent mixture of dimehtyl sulfoxide (10 ml) withdimethylformamide (10 ml) to thereby introduce hydroxysuccinimide intothe polymer chain terminal. Then it was fixed onto an aminopropyl silicagel. The elemental analysis data indicated that the organic mattercontent was increased by 13.1% by weight. This silica gel was packedinto a stainless column. By using a 500 mM aqueous solution of ammoniumsulfate as a mobile phase, insulin β-chain (2 μg) employed as abiological component model was injected into the column. Thus, it wasclarified that the elution time varied depending on temperature.

Example E4

Acrylamide (22 mmol), 3-acrylamide acetanilide (3.9 mmol),3-mercaptopropionic acid (0.3 mmol) and 2,2′-azobis(4-cyanovaleric acid)(0.2 mmol) were dissolved in dimethyl-formamide (14 ml) and subjected toradical polymerization at 70° C. After re-precipitating in ether,poly(acrylamide-co-3-acrylamide acetanilide) was obtained.

This copolymer was dissolved in water and a 300 mM aqueous solution ofsodium chloride each to give a concentration of 1% by weight. Thenchanges in transmittance with temperature change were monitored at 500nm by using a spectrophotometer. FIG. 13 shows the results. In theaqueous solution, the UCST of this copolymer was 25° C. In the 300 mMaqueous sodium chloride solution, the UCST was lowered to 20° C. Thus,it was found that the temperature-responsiveness could be controlleddepending on salt concentration (FIG. 15).

INDUSTRIAL APPLICABILITY

The separation method with the use of the separatory material accordingto the present invention has the following advantages.

1) It is possible to obtain a heat-responsive polymer having a largerside chain than those in the conventional heat-responsive polymerstypified by the amide or ester type polymers.

2) The carbon atom number design and the cloud point can be arbitrarilycontrolled by appropriately combining alkyl groups in the side chain.

3) Thus, bioengineering products (proteins, etc.) having variouspolarities can be separated and purified.

4) The cloud point and the polarity of a polymeric compound can bearbitrarily controlled by appropriately combining two alkyl groups inthe side chain.

In addition, the present invention can provide a temperature-responsivepolymer compound the temperature-responsiveness of which can be easilycontrolled by changing the composition or functional groups of monomersconstituting said polymer compound, the molecular weight of said polymercompound or the concentration of said polymer compound in a solution.Further, the present invention can provide a temperature-responsivepolymer compound having an aromatic ring and being expected as exertinga high hydrophobicity or an electronic interaction which cannot beachieved by the existing temperature-responsive polymer compounds. Thus,it is expected that the present invention can largely contribute to thedevelopment of various temperature-responsive polymer compounds as wellas to the development of adsorption and separation materials containingthese temperature-responsive polymer compounds.

Furthermore, the present invention makes it possible to synthesize aheat-responsive polymer which contains a large amount of hydroxyl groupsand has a highly hydrophobic nature, compared with the conventionalamide-type heat-responsive polymers. Accordingly, the polarity and thehydrogen bonding properties can be controlled over a wide range. Sinethe polarity and the hydrogen bonding properties vary depending on themolecular weight, concentration, density, etc. of the polymer, it isexpected that the polymer compound of the present invention is usable inthe separation, adsorption and release of substance includingbioengineering products.

The present invention also makes it possible to control the expressionof the UCST and the LCST of a polymer in an aqueous solution dependingon the polymer composition. It also makes it possible to express theUCST or the LCST by changing the salt concentration or molecular weight.Moreover, it is found that the expression of the UCST and the LCST canbe controlled. At the same time, the hydrophobic nature and thehydrogen-bonding properties of the polymer in an aqueous solution systemcan be controlled. Taking these effects into consideration, it isexpected that the present invention largely contributes to thedevelopment of techniques for separating, adsorbing and releasingsubstances with the use of temperature-responsive polymer compounds.

1-18. (canceled)
 19. A polymer compound comprising the followingformula:

wherein R represents a linear or branched divalent aliphatic hydrocarbongroup having 1 to 8 carbon atoms, a divalent alicyclic hydrocarbon grouphaving 3 to 8 carbon atoms, or a divalent aromatic hydrocarbon grouphaving 6 to 14 carbon atoms; R′ represents a linear or branchedaliphatic hydrocarbon group having 1 to 8 carbon atoms, a linear orbranched aliphatic hydrocarbon group having one or more hydroxyl groupsand 1 to 8 carbon atoms, a linear or branched aliphatic hydrocarbongroup having one or more acid amide bonds and/or ester bonds and 2 to 9carbon atoms, or a linear or branched aliphatic hydrocarbon group havingone or more acid amide bonds and/or ester bonds, one or more hydroxylgroups and 3 to 9 carbon atoms; and n is an integer of 2 or above;provided that when R represents an methylene group (—CH₂—), R′represents a hydroxymethyl group (—CH₂—OH) or a hyroxyethyl group(—CH₂CH₂—OH).
 20. The polymer compound of claim 19 which has afunctional group at the polymer chain terminal.
 21. The polymer compoundof claim 20, wherein said functional group is selected from the groupconsisting of carboxyl, amino and hydroxyl groups.
 22. The polymercompound of claim 19 which has acid amide bonds at two or more sites inthe polymer side chain.
 23. A heat-responsive polymer material whichcontains a polymer compound represented by the following formula andshows a cloud point due to a temperature change in an aqueous solution:

wherein R represents a linear or branched divalent aliphatic hydrocarbongroup having 1 to 8 carbon atoms, a divalent alicyclic hydrocarbon grouphaving 3 to 8 carbon atoms, or a divalent aromatic hydrocarbon grouphaving 6 to 14 carbon atoms; R′ represents a linear or branchedaliphatic hydrocarbon group having 1 to 8 carbon atoms, a linear orbranched aliphatic hydrocarbon group having one or more hydroxyl groupsand 1 to 8 carbon atoms, a linear or branched aliphatic hydrocarbongroup having one or more acid amide bonds and/or ester bonds and 2 to 9carbon atoms, or a linear or branched aliphatic hydrocarbon group havingone or more acid amide bonds and/or ester bonds, one or more hydroxylgroups and 3 to 9 carbon atoms; and n is an integer of 2 or above;provided that when R represents an methylene group (—CH₂—), R′represents a hydroxymethyl group (—CH₂—OH) or a hyroxyethyl group(—CH₂CH₂—OH).
 24. The heat-responsive polymer material of claim 23,wherein said polymer compound has acid amide bonds at two or more sitesin the polymer side chain.
 25. The heat-responsive polymer material ofclaim 23, wherein the polarity of the hydrophilic nature/hydrophobicnature varies at its cloud point and the polarity of which can becontrolled depending on the pH value, salt concentration or the size ofR and R′.
 26. A Chromatographic packing which contains a polymercompound represented by the following formula and shows a cloud pointdue to a temperature change in an aqueous solution:

wherein R represents a linear or branched divalent aliphatic hydrocarbongroup having 1 to 8 carbon atoms, a divalent alicyclic hydrocarbon grouphaving 3 to 8 carbon atoms, or a divalent aromatic hydrocarbon grouphaving 6 to 14 carbon atoms; R′ represents a linear or branchedaliphatic hydrocarbon group having 1 to 8 carbon atoms, a linear orbranched aliphatic hydrocarbon group having one or more hydroxyl groupsand 1 to 8 carbon atoms, a linear or branched aliphatic hydrocarbongroup having one or more acid amide bonds and/or ester bonds and 2 to 9carbon atoms, or a linear or branched aliphatic hydrocarbon group havingone or more acid amide bonds and/or ester bonds, one or more hydroxylgroups and 3 to 9 carbon atoms; and n is an integer of 2 or above;provided that when R represents an methylene group (—CH₂—), R′represents a hydroxymethyl group (—CH₂—OH) or a hyroxyethyl group(—CH₂CH₂—OH).
 27. The chromatographic packing of claim 26, wherein saidpolymer compound has acid amide bonds at two or more sites in thepolymer side chain.
 28. A method for separating substances comprisingholding a substance on a stationary phase comprising the chromatographicpacking of claim 25, then changing the hydrophilic/hydrophobic balanceof the surface of the stationary phase by a temperature gradient methodwherein the external temperature is changed stepwise, and then passingthrough a single mobile phase, thus effecting separation.
 29. A processfor producing a polymer compound represented by the following formula:

wherein R represents a linear or branched divalent aliphatic hydrocarbongroup having 1 to 8 carbon atoms, a divalent alicyclic hydrocarbon grouphaving 3 to 8 carbon atoms, or a divalent aromatic hydrocarbon grouphaving 6 to 14 carbon atoms; R′ represents a linear or branchedaliphatic hydrocarbon group having 1 to 8 carbon atoms, a linear orbranched aliphatic hydrocarbon group having one or more hydroxyl groupsand 1 to 8 carbon atoms, a linear or branched aliphatic hydrocarbongroup having one or more acid amide bonds and/or ester bonds and 2 to 9carbon atoms, or a linear or branched aliphatic hydrocarbon group havingone or more acid amide bonds and/or ester bonds, one or more hydroxylgroups and 3 to 9 carbon atoms; and n is an integer of 2 or above;provided that when R represents an methylene group (—CH₂—), R′represents a hydroxymethyl group (—CH₂—OH) or a hyroxyethyl group(—CH₂CH₂—OH); characterized by using one of the following methods: (1)reacting a compound selected from among aminoalkyl acrylamide,aminoalkyl methacrylamide, aminoalkyl acrylamide hydrochloride andaminoalkyl methacrylamide hydrochloride with an acid anhydride orlactone, and purifying the thus obtained product followed bypolymerization in a solvent; and (2) reacting an alkyl diamine with anacid anhydride, an alkyl acid chloride or di-t-butyl dicarbonate, orreacting a compound having an amino group and an amide bond in itsmolecule with acryloyl chloride or methacryloyl chloride, and thenpurifying the thus obtained product followed by polymerization in asolvent.
 30. A material for separating or adsorbing biological sampleswhich is a polymer compound represented by the following formula and hasacid amide bonds at two or more sites in the polymer side chain:

wherein R represents a linear or branched divalent aliphatic hydrocarbongroup having 1 to 8 carbon atoms, a divalent alicyclic hydrocarbon grouphaving 3 to 8 carbon atoms, or a divalent aromatic hydrocarbon grouphaving 6 to 14 carbon atoms; R′ represents a linear or branchedaliphatic hydrocarbon group having 1 to 8 carbon atoms, a linear orbranched aliphatic hydrocarbon group having one or more hydroxyl groupsand 1 to 8 carbon atoms, a linear or branched aliphatic hydrocarbongroup having one or more acid amide bonds and/or ester bonds and 2 to 9carbon atoms, or a linear or branched aliphatic hydrocarbon group havingone or more acid amide bonds and/or ester bonds, one or more hydroxylgroups and 3 to 9 carbon atoms; and n is an integer of 2 or above;provided that when R represents an methylene group (—CH₂—), R′represents a hydroxymethyl group (—CH₂—OH) or a hyroxyethyl group(—CH₂CH₂—OH).
 31. The material for separating or adsorbing biologicalsamples of claim 30 which has a functional group at the polymer chainterminal.
 32. The material for separating or adsorbing biologicalsamples of claim 31, wherein said functional group is selected from thegroup consisting of carboxyl, amino and hydroxyl groups.
 33. A methodfor separating substances characterized by comprising holding abiological sample on a stationary phase, then changing thehydrophilic/hydrophobic balance by changing the external temperature andthus adsorbing and separating the biological sample such as cells,wherein said stationary phase contains a polymer compound represented bythe following formula and having acid amide bonds at two or more sitesin the polymer side chain:

wherein R represents a linear or branched divalent aliphatic hydrocarbongroup having 1 to 8 carbon atoms, a divalent alicyclic hydrocarbon grouphaving 3 to 8 carbon atoms, or a divalent aromatic hydrocarbon grouphaving 6 to 14 carbon atoms; R′ represents a linear or branchedaliphatic hydrocarbon group having 1 to 8 carbon atoms, a linear orbranched aliphatic hydrocarbon group having one or more hydroxyl groupsand 1 to 8 carbon atoms, a linear or branched aliphatic hydrocarbongroup having one or more acid amide bonds and/or ester bonds and 2 to 9carbon atoms, or a linear or branched aliphatic hydrocarbon group havingone or more acid amide bonds and/or ester bonds, one or more hydroxylgroups and 3 to 9 carbon atoms; and n is an integer of 2 or above;provided that when R represents an methylene group (—CH₂—), R′represents a hydroxymethyl group (—CH₂—OH) or a hyroxyethyl group(—CH₂CH₂—OH).
 34. The method for separating substances of claim 33,wherein said polymer compound has a functional group at the polymerchain terminal.
 35. The method for separating substances of claim 34,wherein said functional group is selected from the group consisting ofcarboxyl, amino and hydroxyl groups. 36-98. (canceled)